Abstract 210: Development and characterization of a novel immunocompetent mouse model of radioresistant lung cancer

Abstract

Standard treatment for inoperable or unresectable stage I-III NSCLC includes stereotactic ablative radiotherapy (SABR) and concurrent chemo and radiotherapy, but locoregional recurrence occurs in 10%-30% of the patients. In the absence of distant metastasis, re-irradiation is the main curative-intent treatment option available for these patients, but the risk of severe adverse effects and tumor resistance of heavily irradiated patients are challenges to this therapy. On the other hand, the combination between re-irradiation with chemotherapy or immunotherapy is a scenario not extensively studied in the literature. Current existing models have been developed in immunodeficient mice, where the effect of immunotherapy cannot be assessed. To address this issue, we have developed and characterized a lung cancer model of radiation resistance, which can be used in syngeneic mice with fully functional immune system. To this aim, we have generated a mouse adenocarcinoma radioresistant cell line (Lacun3 RR) from previously characterized Lacun3 adenocarcinoma cells. This radioresistant cell line was generated after several rounds of radiation (8 Gy) administered to the parental Lacun3 cells in vitro. Clonogenic and apoptotic assays confirmed that Lacun3 RR were more resistant to radiotherapy than the parental cell line, within the 2-4 Gy range. In addition, in vivo assays performed by subcutaneous injection of these cells showed that Lacun3 RR were completely refractory to 10 Gy irradiation, whereas a significant reduction in tumor volume (~60%) was found for parental Lacun3-derived tumors. Phenotypic characterization of these cells showed that Lacun3 RR cells had increased properties of cancer stem cells (CSC), with significantly higher ability to form tumorspheres. Lacun3 RR cells also showed increased CSC markers (ALDH1, NOTCH1) and slightly lower proliferation rates in vitro than Lacun3 cells. Cell signaling analysis revealed an increase in phosphorylated (p)p38 MAPK and pAKT basal and post-irradiation levels, and lower pH2AX (suggesting less DNA damage) in Lacun3 RR cells. RNAseq and DNAseq (Whole Exome Sequencing) were performed in both cell lines. As expected, we found that a substantial number of genes differentially expressed were significantly related to DNA repair, cell cycle regulation and spindle formation. Mutational burden was estimated from the DNAseq, revealing a similar global tumor mutational burden for both cell lines. However, some mutations were exclusively found in Lacun3 RR cells. The tumor microenvironment and tumor immune populations are currently being investigated in this model. In conclusion, we have developed a mouse model to study radiation resistance in a context of complete immunity, which could be highly valuable to study possible synergistic effects of radiotherapy and immunotherapy of re-irradiated NSCLC patients. Citation Format: Sergio León, Diego Serrano, Esther Redín, Jennifer Barranco, Maeva Houry, Francisco Expósito, Ibón Tamayo, Luis Montuenga, Alfonso Calvo, Jose Javier Aristu. Development and characterization of a novel immunocompetent mouse model of radioresistant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 210.