Assistant Professor Osaka University Suita, Osaka, Japan
Purpose: In order to cure cancer, several methods have been developed over several decades. In terms of drug treatment such as chemotherapy, targeted therapy, and immune checkpoint blockade, the purpose of these drugs is eradicating tumor cells by inducing cell death. Types of cell death are categorized into two major types, programmed cell death and non-programmed cell death. Moreover, programmed cell death are further divided into two types, apoptotic cell death and non-apoptotic programmed cell death. Non-apoptotic cell death consists of autophagy-dependent cell death, pyroptosis, and necroptosis. Among anti-cancer drugs, the majority of them aim to induce apoptotic cell death by inhibiting survival signaling specific to cancer cell. In this point, in the process of anti-cancer drug discovery, new types of cells death, ferroptosis has been proposed in 2012.
Ferroptosis is induced by the accumulation of lipid peroxides, and increased intracellular ferrous ion levels contribute to ferroptosis. Although several chemical compounds have been discovered as a specific inducer of ferroptosis in cancer cells and several studies tried to use these inducers for the purpose of cancer treatment in clinical setting, any ferroptosis inducer has not been approved for clinical use. Considering that tumors are surrounded by specific environment; tumor microenvironment (TME) which compose of tumor cells, blood vessels, immune cells, extracellular matrix, revealing the effect of TME on ferroptosis could be critical issue to precisely understand the mechanisms of ferroptosis induction in tumor cells. Here, we tried to elucidate the mechanisms of ferroptosis induction in cancer by focusing on macrophage which is a one of the major cell types in TME. Methods: Murine triple negative breast cancer line (4T1-luc) and murine macrophage cell line (RAW 264.7) were used in this study. RSL3 was utilized to induce ferroptosis in cancer cells and cell viability was quantified by MTT assay and crystal violet staining. In indirect co-culture experiments, transwell with 0.4 µm pore size was used. Moreover, to evaluate the direct cell-cell interaction, 4T1-luc cells were labeled with green fluorescent protein (GFP) by lentivirus mediated transduction. To capture GFP fluorescence, CV8000 (High content imaging system) was used in this study. Results: To evaluate the effect macrophage on the susceptibility of cancer cells to ferroptosis, we tested three types of experiments which enable us to clarify the important interaction form between cancer cells and macrophages. First, we tested whether secretion from macrophages could affect the sensitivity of cancer cells to ferroptosis inducer. To evaluate the effect of secretion, we collected and used the conditioned medium from macrophages. After incubation of 4T1-luc cells with or without conditioned medium from RAW 264.7 cells, 4T1-luc cells were treated with RSL3 (0, 62.5, 125, 250 nM) for 24 hours and then cell viability was evaluated. As a result, cell viability was not changed due to the incubation with conditioned medium from RAW 264.7 cells. Next, we conducted co-culture experiment by using transwell system to confirm indirect cell-cell interaction via secretion of macrophage and cancer cells. After co-culture of 4T1-luc cells with RAW 264.7 cells by using transwell for 72 hours, RSL3 (0, 62.5, 125, 250 nM) was treated and remaining cells were quantified. Results showed that co-culture with RAW 264.7 cells enhanced the sensitivity of 4T1-luc cells to RSL3 treatment. These findings indicated that interaction with macrophages could be a key factor to determine the ferroptosis susceptibility. Furthermore, we tested the effect of direct cell-cell interaction by co-culturing 4T1-luc cells and RAW 264.7 cells in the same dish. To distinguish two types of cells, 4T1-luc cells were labeled with GFP and co-cultured with RAW 264.7 cells. After treatment of RSL3 (300 nM) on 4T1-luc mono-culture or 4T1-luc and RAW 264.7 cells co-culture, we captured GFP fluorescence by high content imaging system to quantify surviving cancer cells (GFP labeled 4T1-luc cells). As a result, 4T1-luc cells were less in the co-culture condition than mono-culture group after RSL3 treatment, indicating that direct cell-cell interaction is also the determinant of ferroptosis induction. Conclusion: Considering that cancer cells are surrounded by several cell types in TME including macrophages, we tried to evaluate whether macrophages could alter the sensitivity of cancer cells to ferroptosis inducer. We demonstrated that indirect co-culture and direct co-culture of cancer cells (4T1-luc cells) with macrophages (RAW 264.7 cells) enhanced the susceptibility to ferroptosis induction. In order to apply ferroptosis inducers as a therapeutic option for cancer in the future, this findings suggest the importance to understand the mechanisms of ferroptosis induction considering cell-cell interaction in TME.
Acknowledgements: This study was supported by Grant-in-Aid for Research Activity Start-up from the Japan Society for the Promotion of Science.
