In vitro and in vivo YTN16 gastric cancer response to low dose focal SmART+ X-ray irradiation

Background: Prior to 2018, no mouse gastric cancer cell line was implantable in C57BL/6 mice. However, now that this YTN16 mouse cancer model has been established, it can be used for evaluating various therapies in an immunocompetent strain. X-ray radiation therapy is a widely used cancer treatment known to cause DNA damage and dimmish a cells ability to proliferate. The Houston Methodist Research Institute has recently acquired a small animal radiation therapy (SmART+) machine developed by Precision X-ray Irradiation (Madison, CT). This machine is capable of image guided radiation delivery through lower energy computed tomography and 225 kV X-ray irradiation. The machine is designed for use with small animals such as mice, rats and rabbits, but is also capable of irradiation of cells in a dish.

Methods: The response of YTN16 cancer cells to SmART+ radiation therapy was studied in a 96-well plate and in both NSG and C57BL/6 mice. For the in vitro study, focal plane (at isocenter) X-rays were applied to the plate in six distinct quadrants (four by four well grids). The six quadrants of the plate were exposed to X-ray doses of zero (control), two, five, ten, twenty, and forty Gray. Bioluminescence (Tecan Spark) along with bright field and green fluorescent protein microscopy (EVOS M5000) was used to quantify cell survival. For the in vivo study, ten male NSG and ten male C57BL/6 mice were subcutaneously xenografted with one million YTN16 gastric cancer cells per hundred microliters media in a 50% - 50% mixture with Matrigel. Tumors were monitored with a caliper and allowed to grow until they were measured to be one hundred cubic millimeters. Mice were then divided into treatment groups of three mice per cohort with four mice in the control groups. After which, X-ray radiation was applied locally to the tumor in three doses of zero, ten, or twenty Gray. After radiation therapy, one mouse from each cohort was sacrificed and tumors were harvested for tumor microenvironment imaging. The remaining seven mice were monitored for weight loss and tumor shrinkage through scale and caliper measurements.

Results: In the dish, irradiated YTN16 cells showed survival characteristics like other gastric cell cancer lines such as RKO and SW480. In NSG mice, radiation slowed the progression of tumor growth and even caused some shrinkage. In C57BL/6 mice, heightened levels of T-cells and macrophages at the tumor site shown through imaging on the Akoya PhenoCycler show natural immune response to radiation. The level of immune activity was proportional to the radiation dose for the three dose cases explored herein.

Conclusions: Our studies demonstrate that focal SmART+ X-ray radiation can be used to slow or halt YTN16 gastric tumor growth in the petri dish and in immunodeficient and immunocompetent mice. Coupling X-ray radiation with other therapies such a T-cells may have the ability to reduce tumor activity completely.