Correlation of nuclear pIGF-1R/IGF-1R and YAP/TAZ in a tissue microarray with outcomes in osteosarcoma patients.

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.

Transcriptional activators YAP/TAZ and AXL orchestrate dedifferentiation, cell fate, and metastasis in human osteosarcoma.

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.

Modeling the Tumor Microenvironment and Pathogenic Signaling in Bone Sarcoma.

Investigations of cancer biology and screening of potential therapeutics for efficacy and safety begin in the preclinical laboratory setting. A staple of most basic research in cancer involves the use of tissue culture plates, on which immortalized cell lines are grown in monolayers. However, this practice has been in use for over six decades and does not account for vital elements of the tumor microenvironment that are thought to aid in initiation, propagation, and ultimately, metastasis of cancer. Furthermore, information gleaned from these techniques does not always translate to animal models or, more crucially, clinical trials in cancer patients. Osteosarcoma (OS) and Ewing sarcoma (ES) are the most common primary tumors of bone, but outcomes for patients with metastatic or recurrent disease have stagnated in recent decades. The unique elements of the bone tumor microenvironment have been shown to play critical roles in the pathogenesis of these tumors and thus should be incorporated in the preclinical models of these diseases. In recent years, the field of tissue engineering has leveraged techniques used in designing scaffolds for regenerative medicine to engineer preclinical tumor models that incorporate spatiotemporal control of physical and biological elements. We herein review the clinical aspects of OS and ES, critical elements present in the sarcoma microenvironment, and engineering approaches to model the bone tumor microenvironment. Impact statement The current paradigm of cancer biology investigation and therapeutic testing relies heavily on monolayer, monoculture methods developed over half a century ago. However, these methods often lack essential hallmarks of the cancer microenvironment that contribute to tumor pathogenesis. Tissue engineers incorporate scaffolds, mechanical forces, cells, and bioactive signals into biological environments to drive cell phenotype. Investigators of bone sarcomas, aggressive tumors that often rob patients of decades of life, have begun to use tissue engineering techniques to devise in vitro models for these diseases. Their efforts highlight how critical elements of the cancer microenvironment directly affect tumor signaling and pathogenesis.

3D Tissue-Engineered Tumor Model for Ewing's Sarcoma That Incorporates Bone-like ECM and Mineralization.

The tumor microenvironment harbors essential components required for cancer progression including biochemical signals and mechanical cues. To study the effects of microenvironmental elements on Ewing's sarcoma (ES) pathogenesis, we tissue-engineered an acellular three-dimensional (3D) bone tumor niche from electrospun poly(ε-caprolactone) (PCL) scaffolds that incorporate bone-like architecture, extracellular matrix (ECM), and mineralization. PCL-ECM constructs were generated by decellularizing PCL scaffolds harboring cultures of osteogenic human mesenchymal stem cells. The PCL-ECM constructs simulated in vivo-like tumor architecture and increased the proliferation of ES cells compared to PCL scaffolds alone. Compared to monolayer controls, 3D environments facilitated the downregulation of the canonical insulin-like growth factor 1 receptor (IGF-1R) signal cascade through mechanistic target of rapamycin (mTOR), both of which are targets of recent clinical trials. In addition to the downregulation of canonical IGF-1R signaling, 3D environments promoted a reduction in the clathrin-dependent nuclear localization and transcriptional activity of IGF-1R. In vitro drug testing revealed that 3D environments generated cell phenotypes that were resistant to mTOR inhibition and chemotherapy. Our versatile PCL-ECM constructs allow for the investigation of the roles of various microenvironmental elements in ES tumor growth, cancer cell morphology, and induction of resistant cell phenotypes.

Multimaterial Dual Gradient Three-Dimensional Printing for Osteogenic Differentiation and Spatial Segregation.

In this study of three-dimensional (3D) printed composite ß-tricalcium phosphate (ß-TCP)-/hydroxyapatite/poly(ɛ-caprolactone)-based constructs, the effects of vertical compositional ceramic gradients and architectural porosity gradients on the osteogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (MSCs) were investigated. Specifically, three different concentrations of ß-TCP (0, 10, and 20 wt%) and three different porosities (33% ± 4%, 50% ± 4%, and 65% ± 3%) were examined to elucidate the contributions of chemical and physical gradients on the biochemical behavior of MSCs and the mineralized matrix production within a 3D culture system. By delaminating the constructs at the gradient transition point, the spatial separation of cellular phenotypes could be specifically evaluated for each construct section. Results indicated that increased concentrations of ß-TCP resulted in upregulation of osteogenic markers, including alkaline phosphatase activity and mineralized matrix development. Furthermore, MSCs located within regions of higher porosity displayed a more mature osteogenic phenotype compared to MSCs in lower porosity regions. These results demonstrate that 3D printing can be leveraged to create multiphasic gradient constructs to precisely direct the development and function of MSCs, leading to a phenotypic gradient. Impact Statement In this study, three-dimensional (3D) printed ceramic/polymeric constructs containing discrete vertical gradients of both composition and porosity were fabricated to precisely control the osteogenic differentiation of mesenchymal stem cells. By making simple alterations in construct architecture and composition, constructs containing heterogenous populations of cells were generated, where gradients in scaffold design led to corresponding gradients in cellular phenotype. The study demonstrates that 3D printed multiphasic composite constructs can be leveraged to create complex heterogeneous tissues and interfaces.

IGF-1R/mTOR Targeted Therapy for Ewing Sarcoma: A Meta-Analysis of Five IGF-1R-Related Trials Matched to Proteomic and Radiologic Predictive Biomarkers.

Background : Ten to fourteen percent of Ewing sarcoma (ES) study participants treated nationwide with IGF-1 receptor (IGF-1R)-targeted antibodies achieved tumor regression. Despite this success, low response rates and short response durations (approximately 7-weeks) have slowed the development of this therapy. Methods: We performed a meta-analysis of five phase-1b/2 ES-oriented trials that evaluated the anticancer activity of IGF-1R antibodies +/- mTOR inhibitors (mTORi). Our meta-analysis provided a head-to-head comparison of the clinical benefits of IGF-1R antibodies vs. the IGF-1R/mTOR-targeted combination. Available pretreatment clinical samples were semi-quantitatively scored using immunohistochemistry to detect proteins in the IGF-1R/PI3K/AKT/mTOR pathway linked to clinical response. Early PET/CT imaging, obtained within the first 2 weeks (median 10 days), were examined to determine if reduced FDG avidity was predictive of progression-free survival (PFS). Results: Among 56 ES patients treated at MD Anderson Cancer Center (MDACC) with IGF-1R antibodies, our analysis revealed a significant ~two-fold improvement in PFS that favored a combination of IGF-1R/mTORi therapy (1.6 vs. 3.3-months, p = 0.042). Low pIGF-1R in the pretreatment specimens was associated with treatment response. Reduced total-lesion glycolysis more accurately predicted the IGF-1R response than other previously reported radiological biomarkers. Conclusion: Synergistic drug combinations, and newly identified proteomic or radiological biomarkers of IGF-1R response, may be incorporated into future IGF-1R-related trials to improve the response rate in ES patients.

Mechanically tunable coaxial electrospun models of YAP/TAZ mechanoresponse and IGF-1R activation in osteosarcoma.

Current in vitro methods for assessing cancer biology and therapeutic response rely heavily on monolayer cell culture on hard, plastic surfaces that do not recapitulate essential elements of the tumor microenvironment. While a host of tumor models exist, most are not engineered to control the physical properties of the microenvironment and thus may not reflect the effects of mechanotransduction on tumor biology. Utilizing coaxial electrospinning, we developed three-dimensional (3D) tumor models with tunable mechanical properties in order to elucidate the effects of substrate stiffness and tissue architecture in osteosarcoma. Mechanical properties of coaxial electrospun meshes were characterized with a series of macroscale testing with uniaxial tensile testing and microscale testing utilizing atomic force microscopy on single fibers. Calculated moduli in our models ranged over three orders of magnitude in both macroscale and microscale testing. Osteosarcoma cells responded to decreasing substrate stiffness in 3D environments by increasing nuclear localization of Hippo pathway effectors, YAP and TAZ, while downregulating total YAP. Additionally, a downregulation of the IGF-1R/mTOR axis, the target of recent clinical trials in sarcoma, was observed in 3D models and heralded increased resistance to combination chemotherapy and IGF-1R/mTOR targeted agents compared to monolayer controls. In this study, we highlight the necessity of incorporating mechanical cues in cancer biology investigation and the complexity in mechanotransduction as a confluence of stiffness and culture architecture. Our models provide a versatile, mechanically variable substrate on which to study the effects of physical cues on the pathogenesis of tumors. STATEMENT OF SIGNIFICANCE: The tumor microenvironment plays a critical role in cancer pathogenesis. In this work, we engineered 3D, mechanically tunable, coaxial electrospun environments to determine the roles of the mechanical environment on osteosarcoma cell phenotype, morphology, and therapeutic response. We characterize the effects of varying macroscale and microscale stiffnesses in 3D environments on the localization and expression of the mechanoresponsive proteins, YAP and TAZ, and evaluate IGF-1R/mTOR pathway activation, a target of recent clinical trials in sarcoma. Increased nuclear YAP/TAZ was observed as stiffness in 3D was decreased. Downregulation of the IGF-1R/mTOR cascade in all 3D environments was observed. Our study highlights the complexity of mechanotransduction in 3D culture and represents a step towards controlling microenvironmental elements in in vitro cancer investigations.

Effects of Shear Stress Gradients on Ewing Sarcoma Cells Using 3D Printed Scaffolds and Flow Perfusion.

In this work, we combined three-dimensional (3D) scaffolds with flow perfusion bioreactors to evaluate the gradient effects of scaffold architecture and mechanical stimulation, respectively, on tumor cell phenotype. As cancer biologists elucidate the relevance of 3D in vitro tumor models within the drug discovery pipeline, it has become more compelling to model the tumor microenvironment and its impact on tumor cells. In particular, permeability gradients within solid tumors are inherently complex and difficult to accurately model in vitro. However, 3D printing can be used to design scaffolds with complex architecture, and flow perfusion can simulate mechanical stimulation within the tumor microenvironment. By modeling these gradients in vitro with 3D printed scaffolds and flow perfusion, we can identify potential diffusional limitations of drug delivery within a tumor. Ewing sarcoma (ES), a pediatric bone tumor, is a suitable candidate to study heterogeneous tumor response due to its demonstrated shear stress-dependent secretion of ligands important for ES tumor progression. We cultured ES cells under flow perfusion conditions on poly(propylene fumarate) scaffolds, which were fabricated with a distinct pore size gradient via extrusion-based 3D printing. Computational fluid modeling confirmed the presence of a shear stress gradient within the scaffolds and estimated the average shear stress that ES cells experience within each layer. Subsequently, we observed enhanced cell proliferation under flow perfusion within layers supporting lower permeability and increased surface area. Additionally, the effects of shear stress gradients on ES cell signaling transduction of the insulin-like growth factor-1 pathway elicited a response dependent upon the scaffold gradient orientation and the presence of flow-derived shear stress. Our results highlight how 3D printed scaffolds, in combination with flow perfusion in vitro, can effectively model aspects of solid tumor heterogeneity for future drug testing and customized patient therapies.

Modeling Stroma-Induced Drug Resistance in a Tissue-Engineered Tumor Model of Ewing Sarcoma.

Three-dimensional (3D) tumor models are gaining traction in the research community given their capacity to mimic aspects of the tumor microenvironment absent in monolayer systems. In particular, the ability to spatiotemporally control cell placement within ex vivo 3D systems has enabled the study of tumor-stroma interactions. Furthermore, by regulating biomechanical stimuli, one can reveal how biophysical cues affect stromal cell phenotype and how their phenotype impacts tumor drug sensitivity. Both tumor architecture and shear force have profound effects on Ewing sarcoma (ES) cell behavior and are known to elicit ligand-mediated activation of the insulin-like growth factor-1 receptor (IGF-1R), thereby mediating resistance of ES cells to IGF-1R inhibitors. Here, we demonstrate that these same biophysical cues-modeled by coculturing ES cells and mesenchymal stem cells (MSCs) in 3D scaffolds within a flow perfusion bioreactor-activate interleukin-6 and transcription factor Stat3. Critically, an active Stat3 pathway drastically alters the equilibrium of IGF-1R-targeted ligands (IGF-1) and antagonists (IGFBP-3) secreted by MSCs. To elucidate how this might promote ES tumor growth under physiological shear-stress conditions, ES cells and MSCs were co-cultured by using a flow perfusion bioreactor at varying ratios that simulate a wide range of native MSC abundance. Our results indicate that ES cells and MSCs stimulate each other's growth. Co-targeting IGF-1R and Stat3 enhanced antineoplastic activity over monotherapy treatment. Although this discovery requires prospective clinical validation in patients, it reveals the power of employing a more physiological tissue-engineered 3D tumor model to elucidate how tumor cells co-opt stromal cells to acquire drug resistance.

Effects of Local Antibiotic Delivery from Porous Space Maintainers on Infection Clearance and Induction of an Osteogenic Membrane in an Infected Bone Defect.

Reconstruction of large bone defects can be complicated by the presence of both infection and local antibiotic administration. This can be addressed through a two-stage reconstructive approach, called the Masquelet technique, that involves the generation of an induced osteogenic membrane over a temporary poly(methyl methacrylate) (PMMA) space maintainer, followed by definitive reconstruction after the induced membrane is formed. Given that infection and antibiotic delivery each have independent effects on local tissue response, the objective of this study is to evaluate the interaction between local clindamycin release and bacterial contamination with regards to infection prevention and the restoration of pro-osteogenic gene expression in the induced membrane. Porous PMMA space maintainers with or without clindamycin were implanted in an 8 mm rat femoral defect model with or without Staphylococcus aureus inoculation for 28 days in a full-factorial study design (four groups, n = 8/group). Culture results demonstrated that 8/8 animals in the inoculated/no antibiotic group were infected at 4 weeks, which was significantly reduced to 1/8 animals in the inoculated/antibiotic group. Quantitative polymerase chain reaction analysis demonstrated that clindamycin treatment restores inflammatory cytokine and growth factor expression to the same levels as the no inoculation/no antibiotic group, demonstrating that clindamycin can ameliorate the negative effects of bacterial inoculation and does not itself negatively impact the expression of important cytokines. Main effect analysis shows that bacterial inoculation and clindamycin treatment have independent and interacting effects on the gene expression profile of the induced membrane, further highlighting that antibiotics play an important role in the regeneration of infected defects apart from their antimicrobial properties.

Immunomodulatory properties of stem cells and bioactive molecules for tissue engineering.

The immune system plays a crucial role in the success of tissue engineering strategies. Failure to consider the interactions between implantable scaffolds, usually containing cells and/or bioactive molecules, and the immune system can result in rejection of the implant and devastating clinical consequences. However, recent research into mesenchymal stem cells, which are commonly used in many tissue engineering applications, indicates that they may play a beneficial role modulating the immune system. Likewise, direct delivery of bioactive molecules involved in the inflammatory process can promote the success of tissue engineering constructs. In this article, we will review the various mechanisms in which modulation of the immune system is achieved through delivered bioactive molecules and cells and contextualize this information for future strategies in tissue engineering.