Figure 3. Irradiation increases cell migration and induces protumorigenic genes in macrophages.

A) RAW264.7 macrophages were seeded in 8-µm transwell inserts, and tumor conditioned media (collected 48hrs after 3Gy irradiation) was placed in the bottom. Cells were allowed to migrate toward bottom for 6 hrs. Then cells were fixed and stained with DAPI. Representative images of migrated cells are shown and they were quantified using ImageJ software (n=3). B) Effect of irradiation on bone marrow derived macrophages (BMDM). Bone marrows were collected and induced to macrophages by CSF1 (10ng/ml) for 6 days. Cells were counted, seeded and subjected to 3Gy irradiation. Cells were collected 24hrs later, and RT-PCR was performed to detect RNA for the protumorigenic and inflammatory genes noted. C) BMDMs as prepared above were cultured in 50% tumor conditioned media + 50% complete DMEM for 24hrs. Cells were collected and assayed by RT-PCR for the genetic markers noted. D) Tumors collected as shown in Fig 1A were analyzed by FACS for MHCII expression on CD11b⁺F480⁺ macrophages. An increase in MHCII low-expressing macrophage population was observed. (* indicates significant changes with P< 0.05)