(W0930-04-19) Quantitative Phase Imaging for Evaluation of the Radioprotective Effects of Synthetic Glycosaminoglycans on Immune Cells

Purpose: Radiation therapy is a cornerstone of cancer treatment. However, radiation exposure carries the risk of significant side effects due to cellular damage in nonspecific tissues. Developing new strategies to study radiation-damaging effects and how to mitigate radiation-induced toxicities is crucial to enhance cancer therapy outcomes. Quantitative phase imaging (QPI) is a nondestructive, label-free microscopy method used to quantify changes in cellular mass as a function of time. [1] Prophylactic treatment with a synthetic glycosaminoglycan (GAG) (GM-0111) has demonstrated reductions in tissue inflammation and pain in a preclinical model of radiation-induced proctitis. [2] The objective of this study is to employ QPI to determine the radiosensitivity, response time, and potential protective effects of GM-0111 in mast cells upon exposure to irradiation. Mast cells were selected due to their significant contribution to the inflammatory response upon radiation [3]

Methods: Mouse mast cells (P815) were irradiated with an RS 2000 X-ray irradiator (RAD SOURCE Technologies, GA, USA) using the escalating doses listed in Table 1. Irradiated cells were analyzed for changes in mass with respect to irradiation exposure dose at different time points up to 48 hours after radiation using QPI, followed by image processing, by which cells were tracked across different frames. To study the radioprotective effects of GM-111, cells were pretreated with 1.8*10^-1 µM, 1.8 µM, and 18 µM of GM-0111 respectively. The cells were subsequently exposed to 2.5 Gy or 5 Gy X-ray and imaging using differential phase contrast QPI [5, 6]. Total normalized mass was obtained by summing the total measured cell dry mass within each field of view and normalizing by the measured mass at the first imaging time point. Results were compared to those obtained using nonirradiated cells that were also treated with GM-0111.

Results: Our data show that P815 cells are radiosensitive. Upon increasing the radiation exposure, we observe a dose-dependent decrease in cellular mass over time (Figure 1), whereas for nonirradiated cells, the normalized mass increased to approximately 9 times the starting mass. Pretreatment with 1.8*10^-1  mM GM-0111 resulted in higher cellular mass gain compared to the non-treated irradiated cells (Figure 2), suggesting that low concentrations of GM-0111 have radioprotective effects.

Conclusion: Here, we demonstrated the capability of QPI to be used as a method to determine the radiosensitivity of various cell lines and, at the same time, its ability to screen drugs to be used as radioprotective agents prior to radiation therapy. Our data show that P815 cells are radiosensitive and that low concentrations of glycosaminoglycan has the potential to be used as a radioprotective agent. Our results also show that QPI represents a novel, promising tool to study the effects of radiation exposure on different cell lines.

References: 1. Nguyen, T.L., et al., Quantitative Phase Imaging: Recent Advances and Expanding Potential in Biomedicine. ACS Nano, 2022. 16(8): p. 11516-11544.
2. Steinhauff, D., et al., Silk-elastinlike copolymers enhance bioaccumulation of semisynthetic glycosaminoglycan ethers for prevention of radiation induced proctitis. Journal of Controlled Release, 2021. 332: p. 503-515.
3. Blirando, K., et al., Mast cells are an essential component of human radiation proctitis and contribute to experimental colorectal damage in mice. Am J Pathol, 2011. 178(2): p. 640-51.
4. Teresa Pinto, A., et al., Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive and pro-angiogenic capacities. Scientific reports, 2016. 6(1): p. 18765.
5. Tian, L. and L. Waller, Quantitative differential phase contrast imaging in an LED array microscope. Optics express, 2015. 23(9): p. 11394-11403.
6. Moustafa, T.E., et al., Fabrication and validation of an LED array microscope for multimodal, quantitative imaging. HardwareX, 2023. 13: p. e00399.



Fig 1: Normalized P815 cell mass as a function of time after exposure to different radiation doses, analyzed via QPI.



Fig 2: Changes in P815 cell mass over 48 hours following 2.5 Gy radiation in the absence or presence of 1.8*10-1 µM GM-0111.