Effect of STK11 Mutation in the LLC1 Mouse Lewis Lung Anedonacrcinoma Line on Sensitivity to Particle Radiotherapy

Abstract

Objective: Lung adenocarcinoma is a malignant tumor, which is the most common type of non-small cell lung cancer. The low efficiency of standard methods of treatment of lung adenocarcinoma with mutation of the Stk11 tumor suppressor gene is a serious problem in clinical practice. The search for and improvement of new therapeutic approaches to this disease remains an urgent task of modern biomedicine. The aim of the work was to create an in vitro model of lung cancer based on the LLC1 cell line with knockout of the Stk11 gene to assess the sensitivity of mutant cells to various types of radiation therapy, including irradiation with photons, protons and neutrons. Methods: The main methods used were CRISPR/Cas9 genome editing technologies to obtain mutant clones, laser cell sorting, PCR analysis to confirm the deletion, as well as an assessment of the viability, proliferation (metabolic tests, Mki67 marker expression), apoptosis induction (annexin V-PI method), and Pten gene expression after cell irradiation with a dose of 2 Gy. Results and Discussion: As a result, heterozygous mutant lines LLC1-STK11-Mut were obtained. Cell irradiation revealed that in Stk11 mutant cells, radio-induced growth stimulation persisted longer than in wild-type cells, and a significant increase in the proportion of late apoptotic and necrotic cells was observed. At the same time, Mki67 expression temporarily decreased after irradiation, but quickly recovered in mutant cells, which indicates their higher radioresistance. Unlike wild-type cells, the expression level of the Pten gene in mutant cells did not change significantly after irradiation. Conclusions: The Stk11 mutation contributes to the formation of radioresistance in tumor cells by triggering various adaptation mechanisms. The obtained in vitro model can be used for the further study of radioresistance and development of new approaches to the therapy of tumors with the Stk11 mutation.