Cancer growth and invasion reflect many genetic and molecular events. These changes cannot be easily defined in situ, because (a) many factors are difficult to reproduce outside the host and (b) simplifications made to define variables with precision can create artifacts. In this and a prior study  we address a part of this problem. Specifically, we attempt to separate results due to a biological change of interest, the transition from normoxia to hypoxia, from those potentially induced by a simplification of the measurement process, growth in monolayer instead of in three dimensional cultures (3D). We have made other simplifications (e.g., using cell lines as opposed to primary cultures), so we are not perfectly "mimicking" disease conditions. Rather, we are focusing on effects of one specific simplification and outlining an approach that could be used more widely.
The importance of hypoxia to our understanding of tumor growth is based on the premise that all tumors, at some time, exhibit reduced oxygen delivery to the respiring neoplastic and stromal cells. This can be microscopic or macroscopic but can lead to proteome changes in neoplastic and stromal cells leading to impaired neoplastic growth through molecular mechanisms, resulting in cellular quiescence, differentiation, apoptosis, and necrosis [2, 3] and activation of genes, transcription factors, proteins, and cytokine signals that can lead to regional tumor defensive strategies such as angiogenesis, anaerobic glycolysis, locomotion (invasion/metastasis), as well as tumor-specific survival strategies of apoptosis/autophagy [4, 5]. These hypoxia-induced changes have presented challenges for cytotoxic chemotherapy and, likely, will do so for many targeted therapies. In addition, hypoxia diminishes the effectiveness of radiation therapy, in many cases, more for gliomas than for adenocarcinomas [6, 7]. Thus, we hoped that being able to compare and contrast protein and phosphoprotein changes in glioma and adenocarcinoma cells might help design better treatment strategies for gliomas in the future.
The importance of studying protein changes in 3-dimensional (3D) growth is also important since a feature of malignant cells is their ability to grow in 3-dimensions (3D) as spheroids and colonies. This observation has led to greater study of tumors in 3D, as it is closer to in situ growth [8–11] even though it lacks many of the supporting extracellular systems (e.g., endothelial cells and capillaries, supporting matrices, cytokines, etc.). In addition, it has been observed that cancer cell lines grown in 2D and 3D culture respond differently to radiation and cytotoxic drugs [12–14]. Why do cell lines exhibit this differential behavior? Oxygenation of tumor cells also varies with 3D growth as cells grow distant from oxygen and nutrients, whether tumor cells are in 3D culture [15, 16] or part of an in situ tumor [3, 7, 17, 18]. Most studies of hypoxia in tumor cells have utilized 2D cultures [19, 20].
In this study we begin to address the following questions. What protein and phosphoprotein changes reflect adaptations of tumor cells to 3D growth compared to 2D growth? What changes reflect adaptations from normoxia to hypoxia? Do tumor cells from high-grade glioma cell lines respond differently to 3D growth than adenocarcinoma cell lines? When exposed to relative hypoxic (aka microaerophilic) conditions, are changes in protein and phosphoprotein levels more affected by growth in 3D culture than they are by hypoxia?
In this study, we examine levels of 121 phosphorylated and non-phosphorylated proteins using reverse-phase protein array (RPPA)  technology. We examine these levels in eleven cell lines (including both gliomas and adenocarcinomas) under all combinations of media (2D and 3D) and growth conditions (normoxia and hypoxia), allowing us to properly relate changes to causes.