Over the last 40 years, 5 year survival rates for cancer patients have risen dramatically, but total death rates have remained stubbornly high 
. This reflects both our ability to effectively fight primary tumors and our inability to treat secondary or recurring lesions. This clinical failure is mainly due to persistent tumors which arise from resistant populations of cancer cells 
. The molecular mechanisms of chemoresistance have been extensively studied
; however this has not translated into clinical success. The gene expression profile of tumor cells changes after treatment
, leading to a more drug-resistant cell population. This resistant phenotype is initially present in the tumor and is enriched during chemotherapy indicating clonal expansion of the resistant individuals 
. The ability to understand and predict the characteristics of the resistant cells will help increase our ability to treat chemoresistant tumors.
An important aspect of chemoresistance is the role of the tumor microenvironment. Tumor cells exist within a dynamic 3D environment which is not completely replicated by standard in vitro
tissue culture techniques. 2D tissue culture plastic and 3D extracellular matrices (ECM), as scaffolds for cell growth, provide the cell with very different biochemical and mechanical environments. Cells growing in 3D matrices have mechano-transducers and ECM adhesion proteins globally expressed on their surfaces; however, cells grown in 2D only interact with a solid substrate on their basal surface 
. Culturing cells in 2D vs. 3D platforms results in significant changes in cell growth 
, migration 
, morphology 
, and gene expression 
. The influence of matrix composition and mechanics is critically important when investigating cancer cell drug response 
. Previous studies have shown that drug response is heavily influenced by ECM mechanics 
. In addition, patient survival rates can also be linked to the mechanical properties of the surrounding milieu, with a more rigid ECM correlating with poor prognosis 
. These differences contribute to the observation that 2D in vitro
systems are poor indicators of in vivo
. 3D systems therefore provide more clinically relevant results.
Many investigations have shown that ECM interacting proteins can be used as predictors of tumor behavior and patient outcomes
, but few have looked at the composition and characteristics of the matrix itself despite indications from mathematical models that ECM density and homogeneity play critical roles in tumor development 
. This is perhaps due to the difficulty of directly observing and manipulating cells while in biomimetic ECMs in vitro
The phosphatide inositol 3 kinase (PI3K)/AKT pathway is an important intracellular signaling cascade which affects cell growth, migration, protein expression and survival 
. shows a simplified version of this signaling pathway. Elevated PI3K activity is observed in many cancers; either due to a constitutively active PI3K mutant or a loss-of-function PTEN mutant, leading to aggressive cell growth and invasion 
. Several pathway proteins, including PI3K itself and the mammalian target of rapamycin (mTOR) have been investigated as potential targets for new anti-cancer drugs 
. mTOR is a cellular kinase that is found in two distinct complexes 
. Both complexes integrate sensors of nutrient and energy levels and regulate downstream cell functions. Increased mTOR Complex 1 (mTORC1) activity results in higher global protein expression, and inappropriate levels of activity have been linked to several kinds of cancer 
. Phosphorylation of ribosomal protein S6 (pS6) is a downstream indicator of mTORC1 activity 
, whilst phosphorylation of the proline-rich Akt Substrate (PRAS) is an upstream indicator of mTORC1 activity, with PRAS acting to inhibit mTORC1 
. mTOR Complex 2 (mTORC2) regulates cytoskeletal organization and turnover as well as AKT phosphorylation. Increased mTORC2 activity leads to the formation of stress fibers and increased cell mobility 
. Phosphorylation of AKT (pAKT) is a downstream indicator of mTORC2 activity 
. In this study, we employ the dual kinase inhibitor PI103 
. PI103 is a cell-permeable, ATP-competitive inhibitor of both PI3K and mTOR that has been shown to have anti-proliferative affects in various cancer cell lines. PI103 is very potent, with IC50
values on the order of 10–100 nM for various isoforms of mTOR and PI3K 
. It was chosen for this study because U2OS cells express high levels of PI3K/Akt pathway proteins, making them theoretically more susceptible to the effect of PI103. Additionally, PI103 is still being validated as a potential cancer therapy and we believe our 3D culture system is perfectly suited for this type of validation study.
Simplified PI3K/AKT pathway map.
To help understand and quantify the effects of the tumor ECM on cell behavior, we have compared proliferation, migration and protein expression of U2OS osteosarcoma cells between 2D and 3D collagen gels, and investigated the effect of changes in the ECM on inhibition of the PI3K pathway.