Dynamic Study of PDT-Induced Oxidative Stress in Cancer Cells Embedded in 3D Collagen Hydrogel Using Genetically Encoded H2O2-Sensor

Photodynamic therapy (PDT) is a rapidly developing cancer treatment method based on the induction of severe oxidative stress in treated cells. Despite widespread clinical application, the molecular mechanisms underlying the photodynamic reaction have not yet been fully elucidated. Currently, the attention of the scientific community has been drawn to the crucial role of the tumor microenvironment which led to transition from using monolayer cultures of cancer cell to complex 3D in vitro models of tumor growth. Such a transition requires modification of existing methods for assessing cellular viability and metabolic responses to therapeutic interventions. We proposed a method for real-time registration of oxidative stress in response to photodynamic therapy in tumor cells embedded in 3D collagen hydrogel. This approach is based on spectroscopic registration of the integral signal from embedded cells expressing genetically encoded fluorescent sensor. The measuring technique does not require the destruction of the hydrogel and allows real-time recording of cell responses to various types of exposure. Using the genetically encoded HyPer sensor, we registered the wave of the secondary production of H2O2 in PDT treated cells lasting for about 1–2 h after the end of irradiation and demonstrated it transient mode, which add new information about mechanisms of PDT-induced oxidative stress. We believe that the proposed approach can become a potent and cost-effective option for real-time registration of cells’ response to various types of exposure and identification of the underlying mechanisms.