Therapeutic Screening Research Articles

High-throughput formulation of reproducible 3D cancer microenvironments for drug testing in myeloid leukemia.

Leukemic microenvironment has been recognized as a factor that strongly supports the mechanisms of resistance. Therefore, targeting the microenvironment is currently one of the major directions in drug development and preclinical studies in leukemia. Despite the variety of available leukemia 3D culture models, the reproducible generation of miniaturized leukemic microenvironments, suitable for high-throughput drug testing, has remained a challenge. Here, we use droplet microfluidics to generate tens of thousands of highly monodisperse leukemic-bone marrow microenvironments within minutes. We employ gelatin methacryloyl (GelMA) as a model extracellular matrix (ECM) and tune the concentration of the biopolymer, check the impact of other components of the ECM (hyaluronic acid), cell concentration and the ratio of leukemic cells to bone marrow cells within the microbeads to establish the optimal conditions for microtissue formation. We administer model kinase inhibitor, imatinib, at various concentrations to the encapsulated leukemic microtissues, and, via comparing mono- and co-culture conditions (cancer alone vs cancer-stroma), we find that the stroma-leukemia crosstalk systematically protects the encapsulated cells against the drug-induced cytotoxicity. With that we demonstrate that our system mimics the physiological stroma-dependent protection. We discuss applicability of our model to (i) studying the role of direct- or close-contact interactions between the leukemia and bone marrow cells embedded in microscale 3D ECM on the stroma-mediated protection, and (ii) high-throughput screening of anti-cancer therapeutics in personalized leukemia therapies.

Generation of a zebrafish neurofibromatosis model via inducible knockout of nf2a/b.

Neurofibromatosis type 2 (NF-2) is a dominantly inherited genetic disorder that results from variants in the tumor suppressor gene, neurofibromin 2 (NF2). Here, we report the generation of a conditional zebrafish model of neurofibromatosis established by inducible genetic knockout of nf2a/b, the zebrafish homologs of human NF2. Analysis of nf2a and nf2b expression revealed ubiquitous expression of nf2b in the early embryo, with overlapping expression in the neural crest and its derivatives and in the cranial mesenchyme. In contrast, nf2a displayed lower expression levels. Induction of nf2a/b knockout at early stages increased the proliferation of larval Schwann cells and meningeal fibroblasts. Subsequently, in adult zebrafish, nf2a/b knockout triggered the development of a spectrum of tumors, including vestibular Schwannomas, spinal Schwannomas, meningiomas and retinal hamartomas, mirroring the tumor manifestations observed in patients with NF-2. Collectively, these findings highlight the generation of a novel zebrafish model that mimics the complexities of the human NF-2 disorder. Consequently, this model holds significant potential for facilitating therapeutic screening and elucidating key driver genes implicated in NF-2 onset.

Single-cell encoded gene silencing for high-throughput combinatorial siRNA screening.

The use of combinatorial siRNAs shows great promise for drug discovery, but the identification of safe and effective siRNA combinations remains challenging. Here, we develop a massively multiplexed technology for systematic screening of siRNA-based cocktail therapeutics. We employ composite micro-carriers that are responsive to near infrared light and magnetic field to achieve photoporation-facilitated siRNA transfection to individual cells. Thus, randomized gene silencing by different siRNA formulations can be performed with high-throughput single-cell-based analyses. For screening anti-cancer siRNA cocktails, we test more than 1300 siRNA combinations for knocking down multiple genes related to tumor growth, discovering effective 3-siRNA formulations with an emphasis on the critical role of inhibiting Cyclin D1 and survivin, along with their complementary targets for synergic efficacy. This approach enables orders of magnitude reduction in time and cost associated with largescale siRNA screening, and resolves key insights to siRNA pharmacology that are not permissive to existing methods.

Predication on organs-on-chips applications the systematic treatment of breast cancer

Abstract. With the continuous development of cancer therapy, traditional in vitro models have been unable to reproduce the structure of organs and tissues of various parts of the human body, fluid flow and mechanical stimulation characteristics, and the human tumor microenvironment, which is crucial to the study of the behavior and progress of cancer, can not be presented through the animal model in vivo, which shows that the traditional physiological model of cancer can no longer meet the research of human anti-cancer therapy. Benefits from the continuous infusion of cell cultures through the microchannel, the device of organ-on-a-chip improves cell viability and activity and facilitates the simulation of the tumor cell microenvironment, which is expected to further replace traditional zoological experiments. This study reviews a method to simulate the microenvironment of the vitro breast cancer metastasis combined with microfluidic technology, and the application of this device in relevant researches of breast cancer cell metastasis and screening of breast cancer therapeutics.

Tumoroid-On-a-Plate (ToP): Physiologically Relevant Cancer Model Generation and Therapeutic Screening.

Employing three-dimensional (3D) in vitro models, including tumor organoids and spheroids, stands pivotal in enhancing cancer therapy. These models bridge the gap between two-dimensional (2D) cell cultures and complex in vivo environments and offer versatile tools for comprehensive studies into cancer progression, drug responses, and tailored therapies. This study introduces the Tumoroid-on-a-Plate (ToP) device, an innovative ope-surface microfluidic platform designed to create predictive 3D models of solid tumors. The ToP device combines tumor mass, stromal cells, and extracellular matrix (ECM) components, to closely replicate the microenvironment of glioblastoma (GBM) and pancreatic adenocarcinoma (PDAC). Using the advanced ToP model and testing various GBM ECM compositions such as collagen and Rreelin within the model, we can assess how specific elements affect GBM invasiveness. The ToP in vitro model also enables screening chemotherapeutics such as temozolomide and iron-chelators in a single and binary treatment setting on the complex ECM-embedded tumoroids to evaluate their toxicity on GBM and PDAC models viability and apoptosis. Furthermore, co-culturing PDAC tumoroids with human-derived fibroblasts reveals the pro-invasive influence of stromal elements on tumor growth and drug response. This research underscores the value of advanced 3D models like ToP in advancing the understanding of cancer complexity and therapy responses.

DDDR-17. A FACILE HIGH THROUGHPUT DRUG SCREENING PLATFORM ENABLES THE CONSTRUCTION OF A PHARMACO-PROTEOGENOMIC INTERACTION LANDSCAPE IN GLIOBLASTOMA

Abstract Despite decades of intensive research, the number of treatment options for glioblastoma (GBM), a devastating disease, remains very limited. The vast tumor heterogeneity of GBM and the blood-brain barrier (BBB) represent major hurdles to developing effective therapeutics. Here, we present CARPOOL, a high-throughput therapeutic screening approach designed to facilitate accurate and parallel interrogation of numerous patient-derived GBM spheroid cultures (GSCs), capturing the disease’s heterogeneity. We validated CARPOOL in vitro and in vivo and then applied it for drug screening using a library of over 700 BBB-penetrating compounds across 23 omics-profiled GSCs. This screening identified numerous potential anti-GBM compounds, including previously non-oncology drugs. Clustering analysis provided insights into the mechanisms of action of some of the compounds. Coupling these results with omics data of the GSCs enabled the identification of genetic, subtype, and gene expression-based predictors of drug sensitivity. Our study presents a new approach for advancing drug screening and in vivo preclinical therapy evaluation for precision oncology. Furthermore, our construction of a pharmaco-proteogenomic interaction landscape offers a valuable resource for both basic and translational glioblastoma research.

Therapeutic drug monitoring of tenofovir disoproxil and emtricitabine in oral HIV pre-exposure prophylaxis (PrEP) users who have undergone gastrointestinal surgery.

Oral HIV pre-exposure prophylaxis (PrEP) is highly effective in preventing HIV, but its efficacy depends on adequate absorption of drug, which may decrease following gastrointestinal surgery. Clinicians across eight Genito-urinary Medicine clinics in the United Kingdom submitted data on PrEP users with history of gastrointestinal surgery who were referred to a national complex PrEP multi-disciplinary team between June 2021 and April 2023. Anonymised data were submitted on demographics, surgical history, PrEP regimen, and results of therapeutic drug monitoring (TDM) and HIV screening tests. Descriptive analyses were performed in SPSS version 29. Nine cases described cis-gender men-who-have-sex-with-men (MSM) with median age of 47.4 years (IQR = 43 - 56.5) taking tenofovir disoproxil (TDF)/emtricitabine (FTC) daily (n = 8) or event-based (n = 1) as PrEP. Median time between PrEP initiation and TDM was 53 days (IQR = 8.5-1705). The mean (±SD) trough concentration of tenofovir (TFV) and FTC were 90.2 ± 27.7ng/mL and 76.0 ± 45.9ng/mL, respectively. All patients had a negative HIV test at follow-up. Plasma trough concentrations of TFV observed in our cohort taking TDF/FTC were above the expected concentrations associated with PrEP efficacy as previously described in the literature, suggesting that PrEP can be safely given in this population, with TDM used for reassurance.

Enantioselective Synthesis of Chiral Sulfonimidoyl Fluorides Facilitates Stereospecific SuFEx Click Chemistry.

Sulfur-centered electrophilic 'warheads' have emerged as key components for chemical proteomic probes through sulfur-exchange chemistry (SuFEx) with protein nucleophiles. Among these functional groups, sulfonimidoyl fluorides (SIFs) stand out for their modifiable sites, tunable electrophilicities, and chiral sulfur-center, presenting exciting possibilities for new covalent chemical probes. However, the synthetic access to chiral SIFs has been a challenge, limiting their exploration and applications. In this study, we describe a convenient route to obtain chiral SIFs from readily available sulfenamides via a series of one-pot tandem reactions with high enantiomeric excess (ees). The resulting chiral SIFs were further converted into a diverse array of chiral S(VI) derivatives under mild conditions or in buffer solutions. Most significantly, the specificity of the chiral SIFs in protein ligation experiments underscored the critical role of sulfur-center chirality in the design and screening of more-selective covalent probes and therapeutics.

Acoustically tunable intra-droplet assembly of organoids towards high-throughput tumor model construction

Three-dimensional cell organoids and spheroids are increasingly recognized as physiologically relevant models for ex vivo research and therapeutic screening. However, there remains a need for a cost-effective and convenient platform to fabricate these models with well-defined architectures for downstream applications. Here, this work proposes micro models constructing in the droplet platform, by acoustic forces, that can obtain 3D cellular organoids with tunable structures at high yield. By modulating signals applied to acoustic devices, a periodic potential array with adjustable structures is formed, enabling uniform trapping and patterning of droplets. Simultaneously, the localized potential is also formed inside droplets, resulting in the active assembly of cells. Then, hydrogel droplets containing the constructed models are crosslinked, providing ECM to mimic the 3D scaffolds. In experiment, MCF-7 cells are assembled into spherical, linear and cross structures. This method can generate thousands of tumor spheroids in one minute. The long-term culturing results indicate an enhanced efficiency in forming mature tumor models compared with control groups. This biomanufacturing technology has the advantages of being high-throughput, low-cost, and tunable, making it a robust tool for droplet-based applications such as tissue engineering, drug screening, and disease modeling etc.

Long-term cardiovascular outcomes of immune checkpoint inhibitor-related myocarditis: A large single-centre analysis.

Immune checkpoint inhibitors (ICI) are the cornerstone of modern oncology; however, side effects such as ICI-related myocarditis (irM) can be fatal. Recently, Bonaca proposed criteria for irM; however, it is unknown if they correlate well with cardiovascular (CV) ICI-related adverse events. Additionally, whether incident irM portends worse long-term CV outcomes remains unclear. We aimed to determine the incidence of long-term CV comorbidities and CV mortality among irM patients. The ICI-related adverse event (irAE) registry was queried to identify irM patients by using Bonaca criteria. Random controls were selected after excluding patients with other concomitant irAEs. Patients' demographics, comorbidities and myocarditis presenting features were gathered. Outcomes included 2-year freedom from CV comorbidities (composite of atrial fibrillation, stroke, myocardial infarction and heart failure) and freedom from CV death. IrM was treated as a time-varying covariate. Seventy-six patients developed irM at a median of 167days (mean age 69, 63.2% male, 47% lung cancer). Majority of patients had new wall motion abnormalities or EKG changes on presentation. Mean LVEF was 43%, median peak TnT was 0.81, and median NTproBNP was 2057 at irM onset. Two-year freedom from CV comorbidities (67% vs 86.8%, P<0.001) and death (93.4% vs 99.3%, P=0.003) was lower among irM patients. Incident irM was an independent predictor of CV death (HR 8.28, P=0.048), but not CV comorbidities (HR 2.21, P=0.080). This is the largest case-control study on irM highlighting worse long-term CV outcomes. Future studies are needed to establish appropriate therapeutic strategies and efficient screening strategies for irM survivors.

Validation of a functional human AD model with four AD therapeutics utilizing patterned ipsc-derived cortical neurons integrated with microelectrode arrays

Preclinical methods are needed for screening potential Alzheimer’s disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. Long-term potentiation (LTP), which is a correlate of learning and memory, was induced in mature human iPSC-derived cortical neurons cultured on microelectrode arrays utilizing circuit patterns connecting two adjacent electrodes. We demonstrated an LTP system that modeled the MCI and pre-MCI stages of Alzheimer’s and validated this functional system utilizing four AD therapeutics, which was also verified utilizing patch-clamp electrophysiology. LTP was induced by tetanic electrical stimulation, and LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aβ42) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aβ42-induced LTP impairment. The results illustrate the utility of the system as a validated platform to model MCI AD pathology, and potentially for the pre-MCI phase before significant neuronal death. This system also has the potential to become an ideal platform for high-content therapeutic screening for other neurodegenerative diseases.

Three-dimensional silk fibroin scaffolded co-culture of human neuroblastoma and innate immune cells

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It accounts for 50 % of cancers diagnosed in infants less than 1 year old, and 10 % of all pediatric cancer deaths in the United States. High-risk patients have a less than 50 % 5-year survival rate with current treatment strategies. The complex tumor microenvironment of NB makes the development of treatment strategies for high-risk patients challenging. There is increasing evidence that intratumoral immune suppression plays an important role in the progression and invasion of NB tumors. Few three-dimensional (3D) cancer models include components of the innate immune system. This work develops a preclinical 3D NB-immune co-culture model using SK-N-AS NB cells, NK-92 natural killer cells, and THP-1 derived macrophages, co-cultured on porous 3D silk scaffolds to provide tumor architecture. Conditioned media and indirect co-culturing showed changes in SK-N-AS gene expression associated with immunoregulatory signaling, and changes in NK-92 gene expression that are associated with reduced cytotoxicity. This motivated the development of a 3D direct co-culture system in which NB cells were seeded prior to immune cells to allow incorporation and deposition of extracellular matrix within the construct. Immune cells were then incorporated into the model to achieve direct co-culture with SK-N-AS cells. Changes in THP-1 macrophage polarization toward a more M2-like phenotype were observed in 3D direct co-culture, as well as altered NK-92 cell protein secretion and cytotoxic activity. Preliminary testing of immunotherapeutics within the model was conducted on both NB-macrophage and NB-NK co-cultures, but the model demonstrated limited response to immunotherapeutics. This work lays the foundation for building high-throughput therapeutic screening models for the improved treatment NB and other solid tumors.

On-Demand Seizures Facilitate Rapid Screening of Therapeutics for Epilepsy.

Animal models of epilepsy are critical in drug development and therapeutic testing, but dominant methods for pharmaceutical evaluation face a tradeoff between higher throughput and etiological relevance. For example, in temporal lobe epilepsy, a type of epilepsy where seizures originate from limbic structures like the hippocampus, the main screening models are either based on acutely induced seizures in wild type, naïve animals or spontaneous seizures in chronically epileptic animals. Both types have their disadvantages - the acute convulsant or kindling induced seizures do not account for the myriad neuropathological changes in the diseased, epileptic brains, and spontaneous behavioral seizures are sparse in the chronically epileptic models, making it time-intensive to sufficiently power experiments. In this study, we took a mechanistic approach to precipitate seizures "on demand" in chronically epileptic mice. We briefly synchronized principal cells in the CA1 region of the diseased hippocampus to reliably induce stereotyped on-demand behavioral seizures. These induced seizures resembled naturally occurring spontaneous seizures in the epileptic animals and could be stopped by commonly prescribed anti-seizure medications such as levetiracetam and diazepam. Furthermore, we showed that seizures induced in chronically epileptic animals differed from those in naïve animals, highlighting the importance of evaluating therapeutics in the diseased circuit. Taken together, we envision our model to advance the speed at which both pharmacological and closed loop interventions for temporal lobe epilepsy are evaluated.

Controlling Cell Interactions with DNA Directed Assembly.

The creation of complex cellular environments is critical to mimicking tissue environments that will play a critical role in next-generation tissue engineering, stem cell programming, and therapeutic screening. To address this growing need, techniques capable of manipulating cell-cell and cell-material interactions are required that span single-cell to 3D tissue architectures. DNA programmed assembly and placement of cells present a powerful technique for the bottom-up synthesis of living microtissues for probing key questions in cell-cell and cell-material-driven behaviors through its refined control over placement and architecture. This review examines the current state of the art in the programming of cellular interactions with DNA and its applications spanning tissue model building, fundamental cellular biology, and cell manipulation for measurements across a host of applications.

Progression in Near-Infrared Fluorescence Imaging Technology for Lung Cancer Management.

Lung cancer is a major threat to human health and a leading cause of death. Accurate localization of tumors in vivo is crucial for subsequent treatment. In recent years, fluorescent imaging technology has become a focal point in tumor diagnosis and treatment due to its high sensitivity, strong selectivity, non-invasiveness, and multifunctionality. Molecular probes-based fluorescent imaging not only enables real-time in vivo imaging through fluorescence signals but also integrates therapeutic functions, drug screening, and efficacy monitoring to facilitate comprehensive diagnosis and treatment. Among them, near-infrared (NIR) fluorescence imaging is particularly prominent due to its improved in vivo imaging effect. This trend toward multifunctionality is a significant aspect of the future advancement of fluorescent imaging technology. In the past years, great progress has been made in the field of NIR fluorescence imaging for lung cancer management, as well as the emergence of new problems and challenges. This paper generally summarizes the application of NIR fluorescence imaging technology in these areas in the past five years, including the design, detection principles, and clinical applications, with the aim of advancing more efficient NIR fluorescence imaging technologies to enhance the accuracy of tumor diagnosis and treatment.

Clinical Perspectives and Novel Preclinical Models of Malignant Pleural Mesothelioma: A Critical Review.

Pleural mesothelioma (PM), a rare malignant tumor explicitly associated with asbestos and erionite exposures, has become a global health problem due to limited treatment options and a poor prognosis, in which the median life expectancy varies depending on the method of treatment. However, the importance of early diagnosis is emphasized, and the practical methods have not matured yet. This study provides a critical overview of PM, addressing various aspects like epidemiology, etiology, diagnosis, treatment options, and the potential use of advanced technologies like microfluidic chip-based models for research and diagnosis. It initially begins with fundamentals of clinical aspects and then discusses the identification of disease-specific biomarkers in patients' serum or plasma samples, which could potentially be used for early diagnosis. A detailed investigation of the sophisticated preclinical models is highlighted. Recent three-dimensional (3D) model accomplishments, including microarchitecture modeling by transwell coculture, spheroids, organoids, 3D bioprinting constructs, and ex vivo tumor slices, are discussed comprehensively. On-chip models that imitate physiological processes, such as detection chips and therapeutic screening chips, are assessed as potential techniques. The review concludes with a critical and constructive discussion of the growing interest in the topic and its limitations and suggestions.

Reporter Alleles in hiPSCs: Visual Cues on Development and Disease.

Reporter alleles are essential for advancing research with human induced pluripotent stem cells (hiPSCs), notably in developmental biology and disease modeling. This study investigates the state-of-the-art gene-editing techniques tailored for generating reporter alleles in hiPSCs, emphasizing their effectiveness in investigating cellular dynamics and disease mechanisms. Various methodologies, including the application of CRISPR/Cas9 technology, are discussed for accurately integrating reporter genes into the specific genomic loci. The synthesis of findings from the studies utilizing these reporter alleles reveals insights into developmental processes, genetic disorder modeling, and therapeutic screening, consolidating the existing knowledge. These hiPSC-derived models demonstrate remarkable versatility in replicating human diseases and evaluating drug efficacy, thereby accelerating translational research. Furthermore, this review addresses challenges and future directions in refining the reporter allele design and application to bolster their reliability and relevance in biomedical research. Overall, this investigation offers a comprehensive perspective on the methodologies, applications, and implications of reporter alleles in hiPSC-based studies, underscoring their essential role in advancing both fundamental scientific understanding and clinical practice.

Design and construction of a GAP-43 reporter system for potential identification of effective therapeutics for peripheral nerve regeneration

Background: Peripheral nerve injury (PNI) is a condition that can result in muscle paralysis and sensory disturbances. Electrical stimulation and/or the application of exogenous neurotrophic factors and cytokines are effective at enhancing nerve regeneration and is mediated via the expression of regeneration associated genes (RAGs) such as the growth associated protein GAP-43. Therapeutic upregulation of GAP-43 has potential use as a treatment for improving recovery from PNI. Few studies have investigated the potential of increasing GAP-43 for PNI therapeutic purposes and current methods for measuring GAP-43 expression are limited. Aims and Objectives: The broader aim of this work was to construct a motor neuron-like cell model with a GAP-43 reporter system. Such a model would have potential use in screening for novel therapeutics that upregulate GAP-43 and in the optimisation of electrical stimulation treatment in combination with these therapies. The key aim of the work was to design and construct a Cas9 expressing plasmid bearing a gRNA that targets GAP-43 cleavage and a donor plasmid bearing a reporter GFP or Neo cassette flanked with 5’ and 3’ GAP-43 homology arms (HAs) to facilitate the insertion of the reporter immediately 3’ of the GAP-43 promoter via CRISPR/Cas9 homology directed repair (HDR). Such an insertion would enable quantitative measurement of endogenous expression of the GAP-43 gene. Methods and Results: To guide the Cas9 nuclease to the target location, GAP-43 gRNA oligomers were designed and cloned downstream of the U6 promoter in the Cas9 expression plasmid px330, which also expresses the Cas9 gene and the cloned gRNA when transfected into cells. For CRISPR/Cas9 HDR, the 5’ and 3’ GAP-43 HAs were amplified from mouse genomic DNA and cloned into the donor plasmid using Gibson Assembly so that they flanked the reporter cassette. Results: This work successfully constructed the Cas9 gRNA expressing plasmid to target cleavage of the GAP-43 gene in mouse cell lines and has provided the complete design and construction foundation for generation of the reporter system for the endogenous GAP-43 gene in mice.

Embedded Bioprinting of Tumor-Scale Pancreatic Cancer-Stroma 3D Models for Preclinical Drug Screening.

The establishment of organotypic preclinical models that accurately resemble the native tumor microenvironment at an anatomic human scale is highly desirable to level up in vitro platforms potential for screening candidate therapies. The bioengineering of anatomic-scaled three-dimensional (3D) models that emulate native tumor scale while recapitulating their cellular and matrix components remains, however, to be fully realized. In this focus, herein, we leveraged embedded 3D bioprinting for biofabricating pancreatic ductal adenocarcinoma (PDAC) in vitro models combining gelatin-methacryloyl and hyaluronic acid methacrylate extracellular matrix (ECM)-mimetic biomaterials with human pancreatic cancer cells and cancer-associated fibroblasts to generate in vitro models capable of emulating native tumor size (∼6 mm) and stromal elements. By using a viscoelastic continuous polymeric supporting bath, tumor-scale 3D models were rapidly generated (∼50 constructs/h) and easily recovered following in-bath visible light photocrosslinking. As a proof-of-concept, tissue-scale constructs displaying physiomimetic designs were biofabricated. These models also encompass the incorporation of a stromal compartment to better emulate the cellular components of the PDAC native tumor microenvironment (TME) and its stratified spatial organization. Cell-laden tumor-size constructs remained viable for up to 14 days and were responsive to Gemcitabine in a dose-dependent mode. Cancer-stroma models also exhibited increased drug resistance compared to their monotypic counterparts, highlighting the key role of stromal cells in chemotherapeutic resistance. Overall, we report for the first time the freeform biofabrication of PDAC models exhibiting anatomic scale, different structural complexities, and engineered cancer-stromal compartments, being highly valuable for preclinical screening of therapeutics.

Osteoarthritis early-, mid- and late-stage progression in the rat medial meniscus transection model.

Osteoarthritis is a degenerative disease of synovial joints affecting all tissues, including articular cartilage and subchondral bone. Osteoarthritis animal models can recapitulate aspects of human disease progression and are used to test efficacy of drugs, biomaterials, and cell therapies. The rat medial meniscus transection (MMT) model is a surgically induced posttraumatic osteoarthritis model commonly used for preclinical therapeutic screening. We describe herein, the qualitative and quantitative changes to articular cartilage, subchondral bone, and formation of osteophytes at early-, mid-, and late-stages of osteoarthritis progression. Tibia of MMT-operated animals showed proteoglycan loss and fibrillation along articular cartilage surfaces as early as 3-weeks post-surgery. With contrast-enhanced micro-CT technique, quantitative, 3-dimensional analysis of the tibia showed that the articular cartilage thickened at 3- and 6-weeks post-surgery and decreased at 12-weeks post-surgery. This decreased cartilage thickness corresponded with increased lesions in the articular cartilage that led to its full degradation and exposing the subchondral bone layer. Further, subchondral bone thickening was significant at 6-weeks post-surgery and followed cartilage damage. Osteophytes were found as early as 3-weeks post-surgery and coincided with articular cartilage degradation. Cartilaginous osteophytes preceded mineralization, suggesting endochondral ossification. The rat MMT model has predominantly been used out to 3-weeks, and most studies determined the effect of therapies to delay or prevent the onset of osteoarthritis. We provide evidence that an extension of the rat MMT model out to 6- and 12-weeks more resembled severe phenotypes of human osteoarthritis. Thus, evaluating novel therapeutics at late-stage will be important for eventual clinical translation.