Poster S9 - Quantifying the dynamics of the cell cycle regulator p21 in response to DNA damage in cancer spheroids

Introduction: Cancer treatments that induce DNA damage, such as radiation therapy, rely on the premise that cancer cells cannot properly repair damaged DNA and therefore are unable to continue proliferating. A key regulator arresting the cell cycle and allowing time for proper DNA repair is the gene CDKN1A, encoding the protein p21, a cyclin dependent kinase inhibitor. Studies in monolayer 2-dimensional (2-D) cultured cells have revealed that p21 dynamics following DNA damage depend on the cell cycle; cells in S phase showed minimal p21 accumulation, while cells in G1 and G2 showed a continuous increase in a stepwise manner. The derivatives of p21 protein levels of G1 and G2 cells have shown oscillating rates of p21 protein accumulation, reflecting both protein production and degradation. 2-D cultures lack many of the factors found in tumors, including cell–cell interactions and heterogeneous access to nutrients and oxygen, leading to metabolically distinct zones. Such factors are better modeled in 3-dimensional (3-D) systems (spheroids), which more accurately resemble an in vivo environment. However, there are currently few experimental or computational tools to properly study the dynamics of signaling molecules in 3-D configurations at the single cell level.

Materials and

Methods: In this study, we established spheroid cultures of human breast cancer cells expressing fluorescently tagged p21 and a nuclear marker. We induced DNA damage using irradiation and embedded the spheroids between two thin layers of Matrigel, prior to imaging with a light sheet microscope. As a control, we imaged individually seeded cells under similar Matrigel conditions and following irradiation. In addition, we developed new computational tools for the analysis of p21 dynamics in both individually seeded cells and individual cells within spheroids.

Results, Conclusions, and Discussions: I will present statistical analyses of p21 activation and dynamics obtained from these measurements and their heterogeneities between individual cells in 3-D configurations compared to their known dynamics in 2-D. The results from this study provide a novel physiological platform, as well as computational approaches, for investigating the behavior of other key signaling molecules responding to DNA damage in spheroids at the single cell level.

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