Background
Most advanced non-small cell lung cancer (NSCLC) patients will receive radiotherapy (RT) during the course of their treatment. However, response to RT varies widely among patients and radioresistance remains a major impediment to cure. Despite the addition of immune check point inhibitors to RT, many patients have inherent resistance or develop resistance after such treatment. There’s a significant unmet need to improve RT treatment in lung cancer. In order to deliver personalized RT treatment, it’s important to gain an understanding of the mechanisms that lead to radioresistance. In this work, we identified toll like receptor 4 (TLR4) as a potential target to improve response to RT. Our preliminary data shows that radiation increases TLR4 expression leading to increased regulatory T cell infiltration, tumor cell proliferation and migration. We therefore hypothesized that inhibition of TLR4 signalling can lead to decreased tumor growth and promote infiltration of cytotoxic T cells.
Methods
We used two murine NSCLC cell lines, adenocarcinoma (LLCI) and squamous cell carcinoma (KLN205). Cells were irradiated at 2 Gy and 10 Gy. TLR4 expression was evaluated by qPCR and Western blot. Migration, MTT and crystal violet staining assays were performed with and without the TLR4 inhibitor, TAK242. Apoptosis was evaluated using Annexin V staining. For in vivo experiments, KLN205 cells were inoculated into DBA/2 mice and divided into 4 groups: control, RT, TAK242 and TAK242+RT. Tumour volume was measured using callipers. Flow cytometry was performed to assess the impact of treatment on the tumor immune microenvironment.
Results
Both LLCI and KLN205 cell lines showed increased gene expression of TLR4 and MyD88 in response to 2 Gy and a further increase at 10 Gy in vitro. KLN205 showed similar results in vivo. At the protein level, radiation induced TLR4 and activated the downstream MyD88 pathway for the KLN205 cell line. TLR4 inhibition increased apoptosis and reduced cell proliferation and migration. In vivo, the combination of TLR4 inhibition and RT significantly reduced tumour growth and regulatory T cell infiltration.
Conclusions
Based on these preliminary results, we suggest that radiation induces TLR4 expression leading to increased migration and proliferation of NSCLC cell lines. Inhibition of TLR4 in combination with RT is a promising strategy to improve RT response.
Ethics Approval
Animal studies were approved by the institutional ethics committee of Université de Sherbrooke, Sherbrooke (protocol 363–14).