Browsing by Author "Ludwig, Joseph A."
Now showing 1 - 6 of 6
Results Per Page
Sort Options
Item 3D tissue-engineered model of Ewing's sarcoma(Elsevier, 2014) Lamhamedi-Cherradi, Salah-Eddine; Santoro, Marco; Ramammoorthy, Vandhana; Menegaz, Brian A.; Bartholomeusz, Geoffrey; Iles, Lakesla R.; Amin, Hesham M.; Livingston, J. Andrew; Mikos, Antonios G.; Ludwig, Joseph A.Despite longstanding reliance upon monolayer culture for studying cancer cells, and numerous advantages from both a practical and experimental standpoint, a growing body of evidence suggests that more complex three-dimensional (3D) models are necessary to properly mimic many of the critical hallmarks associated with the oncogenesis, maintenance and spread of Ewing's sarcoma (ES), the second most common pediatric bone tumor. And as clinicians increasingly turn to biologically-targeted therapies that exert their effects not only on the tumor cells themselves, but also on the surrounding extracellular matrix, it is especially important that preclinical models evolve in parallel to reliably measure antineoplastic effects and possible mechanisms of de novo and acquired drug resistance. Herein, we highlight a number of innovative methods used to fabricate biomimetic ES tumors, encompassing both the surrounding cellular milieu and the extracellular matrix (ECM), and suggest potential applications to advance our understanding of ES biology, preclinical drug testing, and personalized medicine.Item Co-Cultures of Articular Chondrocytes and Mesenchymal Stem Cells for Cartilage Tissue Engineering(2014-04-24) Dahlin, Rebecca L.; Mikos, Antonios G.; Kasper, Kurt; Ludwig, Joseph A.; Zygourakis, KyriacosArticular cartilage lines the surfaces of synovial joints to protect underlying bone and provide a smooth surface for articulation. Damage to articular cartilage typically leads to long-term pain and disability, as current treatments are unable to fully restore the functional tissue. Thus, tissue engineers seek to develop technologies to enhance cartilage repair. This thesis investigated two strategies for cartilage engineering: flow perfusion bioreactor culture and co-cultures of chondrocytes with mesenchymal stem cells (MSCs). First, we designed a novel bioreactor and then investigated the effect of flow perfusion on chondrocytes when combined with chondrogenic stimuli, including hypoxia and transforming growth factor-β3 (TGF-β3). We demonstrated that the combination of flow perfusion and hypoxic conditions enhanced proliferation, cartilage-like extracellular matrix production, and chondrogenic gene expression compared to perfusion alone. However, these results also demonstrated the need for a more potent chondrogenic stimulus, and thus the effect of perfusion with TGF-β3 was investigated on both chondrocytes and co-cultures of chondrocytes and MSCs. Here, we described the advantages of using exogenous growth factors in flow perfusion cultures, and the utility of flow perfusion for creating large tissue-engineered constructs. The second part of this thesis investigated co-cultures of chondrocytes and MSCs having the potential to reduce the demand for chondrocytes, which overcomes a significant challenge to current approaches toward cartilage repair. We first investigated the sensitivity of this cell population to TGF-β3 and then investigated the stability of the cell phenotype resulting from growth factor supplementation. The results demonstrated that co-cultures of chondrocytes and MSCs enable a reduced concentration and duration of TGF-β3 exposure to achieve an equivalent level of chondrogenesis compared to chondrocyte or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-β3. The final section of this thesis investigated the ability of such co-cultures to repair cartilage in a rat osteochondral defect model. Here, it was demonstrated that co-cultures achieved equivalent cartilage repair compared to the chondrocytes, thus demonstrating the potential use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.Item Correlation of nuclear pIGF-1R/IGF-1R and YAP/TAZ in a tissue microarray with outcomes in osteosarcoma patients(Oncotarget, 2022) Molina, Eric R.; Chim, Letitia K.; Lamhamedi-Cherradi, Salah-Eddine; Mohiuddin, Sana; McCall, David; Cuglievan, Branko; Krishnan, Sandhya; Porter, Robert W.; Ingram, Davis R.; Wang, Wei-Lien; Lazar, Alexander J.; Scott, David W.; Truong, Danh D.; Daw, Najat C.; Ludwig, Joseph A.; Mikos, Antonios G.Osteosarcoma (OS) is a genetically diverse bone cancer that lacks a consistent targetable mutation. Recent studies suggest the IGF/PI3K/mTOR pathway and YAP/TAZ paralogs regulate cell fate and proliferation in response to biomechanical cues within the tumor microenvironment. How this occurs and their implication upon osteosarcoma survival, remains poorly understood. Here, we show that IGF-1R can translocate into the nucleus, where it may act as part of a transcription factor complex. To explore the relationship between YAP/TAZ and total and nuclear phosphorylated IGF-1R (pIGF-1R), we evaluated sequential tumor sections from a 37-patient tissue microarray by confocal microscopy. Next, we examined the relationship between stained markers, clinical disease characteristics, and patient outcomes. The nuclear to cytoplasmic ratios (N:C ratio) of YAP and TAZ strongly correlated with nuclear pIGF-1R (r = 0.522, p = 0.001 for each pair). Kaplan–Meier analyses indicated that nuclear pIGF-1R predicted poor overall survival, a finding confirmed in the Cox proportional hazards model. Though additional investigation in a larger prospective study will be required to validate the prognostic accuracy of these markers, our results may have broad implications for the new class of YAP, TAZ, AXL, or TEAD inhibitors that have reached early phase clinical trials this year.Item Development of a 3D Tissue Engineered Bone Tumor Model(2013-09-16) Burdett, Emily; Mikos, Antonios G.; Ludwig, Joseph A.; Kasper, Kurt; Jacot, Jeffrey G.; Zygourakis, Kyriacos3D ex vivo tumor models are required which better replicate the microenvironment encountered by tumor cells in vivo. In this study, we applied bone tissue engineering culture techniques to develop an ex vivo 3D bone tumor model. Ewing sarcoma cells were cultured on poly(ε-caprolactone) (PCL) microfiber scaffolds, and cellular growth kinetics, morphology, and infiltration were assessed. Cell/scaffold constructs were then exposed to anticancer drugs for up to 16 days and drug response was compared to 2D controls. Ewing sarcoma cells were capable of attachment and proliferation on PCL scaffolds and dense scaffold infiltration up to 200 micrometers. Constructs could be maintained in culture for up to 32 days, and high density 3D cell growth conferred an increased resistance to anticancer drugs over 2D controls. This 3D tumor model shows potential for use in future studies of bone tumor biology, especially as it pertains to the development of new anticancer drugs.Item Polymer-Based Approaches to Bone Tissue Repair and Regeneration(2014-04-03) Henslee, Allan; Kasper, F. Kurtis; Mikos, Antonios G.; Zygourakis, Kyriacos; Wong, Mark E.; Ludwig, Joseph A.; Bennett, George N.The loss or damage of bone tissue due to trauma or surgical resection remains a significant clinical challenge. Limitations associated with the current gold standard of care, autografting, include donor site morbidity and an inherent lack of availability; thus prompting the need for alternative therapies and materials. Poly(propylene fumarate) (PPF) is a synthetic polymer that has previously been explored for bone tissue engineering applications. In this work, the properties of various formulations of PPF were optimized for specific clinical applications. Initially, a composite scaffold comprised of a solid PPF intramedullary rod and porous sleeve was evaluated in a segmental rat femoral defect model. The presence of the scaffold was able to increase the mechanical stability of the defect but also may have acted as a physical barrier to bone formation. In accordance with these results and through interactions with clinical collaborators, subsequent formulations of PPF targeted bony fractures and defects of the mandible. Key formulation parameters of PPF were varied in order to develop constructs with handling and mechanical properties suitable for mandibular fracture repair applications. Further consideration of the clinical relevancy of PPF-based materials led to the development of PPF as an analog to FDA-approved poly(methyl methacrylate) (PMMA)-based bone cement products. It was found that introducing crosslinked PPF particles to a liquid PPF phase could reduce the maximum crosslinking temperature as well as produce setting and handling characteristics comparable to that of current PMMA-based products. Lastly, as the development of biomaterials must continually adapt to fulfill changing clinical needs, a clinically focused project was performed in which porous PMMA-based implants were fabricated, characterized, and implanted under physician direction. Although successful, the limitations of PMMA including its non-degradable nature and the potential for bacterial contamination led to the development of degradable, porous PPF constructs capable of local antibiotic delivery for craniofacial applications. Properties of interest including degradation, porosity change over time, and antibiotic release kinetics were found to be suitable for craniofacial applications. The studies described here showcase the range of clinical applications that may be fulfilled by PPF-based materials.Item Transcriptional activators YAP/TAZ and AXL orchestrate dedifferentiation, cell fate, and metastasis in human osteosarcoma(Springer Nature, 2021) Lamhamedi-Cherradi, Salah-Eddine; Mohiuddin, Sana; Mishra, Dhruva K.; Krishnan, Sandhya; Velasco, Alejandra Ruiz; Vetter, Amelia M.; Pence, Kristi; McCall, David; Truong, Danh D.; Cuglievan, Branko; Menegaz, Brian A.; Utama, Budi; Daw, Najat C.; Molina, Eric R.; Zielinski, Rafal J.; Livingston, John A.; Gorlick, Richard; Mikos, Antonios G.; Kim, Min P.; Ludwig, Joseph A.Osteosarcoma (OS) is a molecularly heterogeneous, aggressive, poorly differentiated pediatric bone cancer that frequently spreads to the lung. Relatively little is known about phenotypic and epigenetic changes that promote lung metastases. To identify key drivers of metastasis, we studied human CCH-OS-D OS cells within a previously described rat acellular lung (ACL) model that preserves the native lung architecture, extracellular matrix, and capillary network. This system identified a subset of cells—termed derived circulating tumor cells (dCTCs)—that can migrate, intravasate, and spread within a bioreactor-perfused capillary network. Remarkably, dCTCs highly expressed epithelial-to-mesenchymal transition (EMT)-associated transcription factors (EMT-TFs), such as ZEB1, TWIST, and SOX9, which suggests that they undergo cellular reprogramming toward a less differentiated state by coopting the same epigenetic machinery used by carcinomas. Since YAP/TAZ and AXL tightly regulate the fate and plasticity of normal mesenchymal cells in response to microenvironmental cues, we explored whether these proteins contributed to OS metastatic potential using an isogenic pair of human OS cell lines that differ in AXL expression. We show that AXL inhibition significantly reduced the number of MG63.2 pulmonary metastases in murine models. Collectively, we present a laboratory-based method to detect and characterize a pure population of dCTCs, which provides a unique opportunity to study how OS cell fate and differentiation contributes to metastatic potential. Though the important step of clinical validation remains, our identification of AXL, ZEB1, and TWIST upregulation raises the tantalizing prospect that EMT-TF-directed therapies might expand the arsenal of therapies used to combat advanced-stage OS.