Gamma-ray irradiation differentially modulates PD-1 and CTLA-4 expression and tumour growth in parental and acquired radioresistant EMT6 mouse models

Background: Immune checkpoint proteins such as PD-1 and CTLA-4 play pivotal roles in tumour immune evasion. Our previous in vitro studies demonstrated upregulation of Pdcd1 and Ctla4 mRNA in the acquired radioresistant murine breast cancer cell line EMT6RR_MJI. This study aimed to evaluate their gene expression in vivo and assess the impact of gamma-ray irradiation on tumour progression. Methods: Two in vivo experiments were conducted using a mouse xenograft model subcutaneously implanted with either parental EMT6 or EMT6RR_MJI mammary carcinoma cells. In Experiment 1, levels of Pdcd1, Cd274, and Ctla4 mRNA were quantified by real-time PCR from tumours relative to control groups in both models. Mice in both control and treated groups were sacrificed on day 19 post-inoculation (5 days post-irradiation for treated groups), and tumour origin was validated by determining the expression of epithelial marker E-cadherin (Cdh1) and mesenchymal marker N-cadherin (Cdh2). In Experiment 2, tumour volume was measured weekly to assess treatment response relative to controls. Mice were sacrificed if they lost ≥10% of their body weight or showed signs of stress or ulceration. Results: Pdcd1 expression was significantly higher in EMT6RR_MJI tumours compared to parental EMT6 tumours (p