Tissue-Engineered Tumor Microenvironments for Bone Sarcoma
Molina, Eric Rodolfo
Mikos, Antonios G
Doctor of Philosophy
Preclinical methods interrogation of cancer biology and of evaluation of potential therapeutics rely heavily on the use of monolayer, adherent culture on tissue culture treated polystyrene dishes. However, these techniques ignore characteristics of the tumor microenvironment that have been found responsible for essential elements of pathogenesis in tumors. While a variety of in vitro tumor models exist and mouse models with patient derived xenograft (PDX) tissue remain the gold standard in preclinical testing, there is more investigation needed into how various physical and biochemical elements of the microenvironment contribute to cancer pathogenesis. Osteosarcoma (OS) and Ewing’s sarcoma (ES) are the two most common primary tumor of bone. Although advancements in combination therapy and surgical resection have improved outcomes, identification and targeting of essential pathogenic signaling has been difficult as evidenced by myriad failed clinical trials and the absence of biologically targeted therapeutics for these diseases. In this thesis, we employ the techniques of tissue engineering to develop bone tumor microenvironments suitable for the interrogation of elements in sarcoma tumor niche. We sought to determine how these elements contribute to changes in cell phenotype, proliferation, activation of critical pathogenic pathways, and response to therapy. The first objective of this thesis was to engineer a 3D electrospun poly(ε-caprolactone) (PCL) microenvironment to determine the effects of bone-like extracellular matrix (ECM) and mineral components on ES cells. We achieved this by culturing mesenchymal stem cells in osteogenic medium on electrospun scaffolds PCL scaffolds and decellularizing the scaffold after 12 days to generate PCL-ECM constructs. In our first specific aim we show that 3D microenvironments contribute to decreased proliferation and the downregulation of the insulin-like growth factor 1(IGF-1R)/mechanistic target of rapamycin signaling (mTOR) cascade compared to monolayer controls. Further we determine that cells in 3D environments also became resistant to combination therapy with doxorubicin and IGF-1R/mTOR targeted therapy. While the ECM and mineral components in 3D scaffolds increased growth and recapitulated some morphological aspects characteristic of ES tumors in patients, there did not appear to be any difference in IGF-1R/mTOR activation or therapeutic response between ES cultured in either electrospun PCL scaffolds or PCL-ECM constructs. Our second objective was to engineer a mechanically tunable 3D electrospun environment that could be used to interrogate the effects of architecture and stiffness on osteosarcoma. In our second aim, we develop a system of 3D mechanically tunable scaffolds utilizing coaxial electrospinning of PCL:gelatin (core:shell) fibers. We determine by quantitative confocal image analysis that as stiffness is decreased in 3D microenvironments, OS cells increase the nuclear to cytoplasmic ratio (N:C ratio) of the mechanoresponsive proteins, yes-associated protein 1 (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). We further determine that 3D microenvironments contribute to a downregulation of the IGF-1R/mTOR cascade and resistance to combination therapy with chemotherapy and IGF-1R/mTOR targeted therapy compared to monolayer controls in OS. Our third objective was to correlate YAP N:C ratio, TAZ N:C ratio, nuclear IGF-1R, and nuclear phosphorylated IGF-1R (pIGF-1R) in tumor sections from 37 osteosarcoma patients with clinical aspects of disease, histological characterization of tumors, and overall survival. We determined that strong correlations exist between YAP and TAZ N:C ratio and IGF-1R nuclear staining intensity and pIGF-1R staining intensity. Interestingly we also found that pIGF-1R nuclear staining strongly correlated with YAP and TAZ N:C ratio. We determine that nuclear pIGF-1R and YAP N:C ratio were higher in tumors with a chondroblastic histotype and that Nuclear pIGF-1R, YAP N:C ratio, and TAZ N:C ratio were lower in high grade bone osteosarcoma subtypes compared to all other subtypes. Univariate and multivariate analysis of outcomes indicated that high pIGF-1R and possibly low YAP N:C ratio may be negative prognostic indicators for overall survival. The overall goal of this work was to examine how the microenvironment can be engineered to modulate and study essential pathogenic signaling and the generation of therapy resistant phenotypes in sarcoma. Through this thesis, we have demonstrated that architectural elements and the stiffness of tumor environments contribute to phenotypic changes, herald increased therapy resistance in vitro, and affect the expression and localization of potentially prognostic indicators in sarcoma. We highlight the importance of incorporating controllable microenvironmental elements in in vitro cancer biology which can be engineered to study myriad aspects of the tumor microenvironment and recapitulate more accurately the phenotypes of cells found in tumors from patients.
Tumor model; Tissue-Engineering; Osteosarcoma; Ewing's sarcoma; Tumor microenvironment