Background
Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability. In preclinical models, TTFields have been shown to induce cancer immunogenic cell death and elicit a systemic anti-cancer immune response. In a phase 3 study, TTFields co-application with an immune checkpoint inhibitor (ICI) demonstrated benefit in patients with non-small cell lung carcinoma. The current research examined possible effects of TTFields on macrophage phenotype regulation.
Methods
Bone marrow–derived macrophages (BMDMs) were generated by flushing bone marrow from the femurs and tibias of 8–10-week-old BalbC or C57bl/6 mice. Bone marrow cells were cultured in the presence of granulocyte-colony stimulating factor (GM-CSF) for 7 days. Subsequently, to induce M1 polarization, BMDMs were exposed to either IFN- alone or IFN- with LPS. To induce M2 polarization, BMDMs were treated with IL-4. The polarized BMDMs were then treated for 24 hours with TTFields (150 kHz), followed by flow cytometry examination of surface expression of the F4/80 macrophage biomarker and the activation markers: CD80, major histocompatibility complex class II (MHC II), inducible nitric oxide synthase (iNOS), CD206, and ARG-1. Macrophage killing assay was performed by introducing pancreatic cancer cells (Panc02-luc) to the culture of the TTFields-treated macrophages. After 24 hours of co-incubation, the viability of the cancer cells was evaluated by adding luciferin and measuring the bioluminescence signal. Lysates of TTFields-treated macrophages were examined for RhoA activation using an appropriate kit and by Western blot phosphorylation of GEF-H1, c-Jun, and the NFB subunit p65.
Results
Following TTFields exposure, BMDMs demonstrated elevated expression of CD80+, and MHC IIhigh pro-inflammatory M1 markers, and decreased expression of CD206 and ARG-1 M2 markers. iNOS expression was markedly elevated following TTFields treatment to BMDMs that were exposed to IFN-, to a level similar to IFN- plus LPS exposure. BMDMs exposed to TTFields induced killing of pancreatic cancer cells, as suggested by the decreased bioluminescent luciferase signal emitted by the Panc02 cells. TTFields treatment induced activation of RhoA, and phosphorylation of GEF-H1, c-Jun and p65 in macrophages.
Conclusions
This study reveals a novel immunomodulatory role for TTFields, promoting in vitro pro-inflammatory macrophage polarization and activation. TTFields exhibit LPS-like activity by acting as a second signal to polarize M1 macrophages possibly via transcriptional response driven by the GEF-H1/RhoA/ROCK/NFB pathway.