Tumor-Treating Fields Induce RAW264.7 Macrophage Activation Via NK-?B/MAPK Signaling Pathways

Park, Jeong-In; Song, Kyung-Hee; Jung, Seung-Youn; Ahn, Jiyeon; Hwang, Sang-Gu; Kim, Joon; Kim, Eun Ho; Song, Jie-Young

Among the various cytokines, TNF-a can activate macrophages in an autocrine manner to induce the expression of other inflammatory and immunomodulatory mediators. 16 RAW 264.7 cells were treated with TTFs or LPS for 24 hours, and the mRNA expression levels of proinflammatory cytokines such as IL-1b and TNF-a were determined ( Figure 3A) . The mRNA levels of IL-1b and TNF-a were significantly upregulated by the administration of TTFs, and further increase was observed in LPS-activated RAW 264.7 cells. We further investigated the release of some key proinflammatory cytokines in the coculture medium. Compared with that in other groups, IL-1b, TNF-a, and IL-6 levels in the coculture of TTFs-treated RAW 264.7 cells with 4T1 cells were significantly increased at 48 hours in a manner dependent on the ratio of the 2 cells ( Figure 3B ). To evaluate the cytotoxic activity of the CM of TTFs-treated RAW 264.7 cells against 4T1 cells, an MTT assay was performed. As shown in Figure 3C , 4T1 cells exposed to the CM of unstimulated RAW 264.7 cells did not show statistically significant decrease in viability, whereas 4T1 cells exposed to CM from TTFs-treated RAW 264.7 cells exhibited decreased viability after 24 or 48 hours contact with the CM. As expected, the CM of LPS-treated RAW 264.7 cells also significantly decreased the viability of 4T1 cells. These results suggested that TTFs activate macrophages and promote the expression of proinflammatory cytokines.