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. 2023 Nov 13;11(11):e006677. doi: 10.1136/jitc-2023-006677

Figure 4.

Figure 4

Interleukin (IL)-6 secretion by glioblastoma cells defined the tumor-initiating capacity in immunocompetent animals. (A) Cytokine secretion in 8B and 9G cells and medium was analyzed by cytokine array. Each dot represents the cytokine concentration by three independent experiments. (B) Identification of the responsible cytokine(s) secreted by 8B that induce macrophages (Mφs) into a senescence-like state. Splenocytes were cultured in medium containing recombinant cytokines corresponding to the types of cytokines secreted by 8B identified in A. Glioma medium as used for 8B or 9G cultures was used to culture splenocytes. To support Mφs survival, macrophage colony stimulating factor (M-CSF) and IL-34 were added to the glioma medium (base-medium). To determine the factors responsible for Mφ senescence, Base-medium containing the major factors secreted by 8B, including Ccl2, Cxcl1, Cxcl10, Ccl5, and IL-6 (all factors) was used. Each factor from a pool of five cytokines were removed from the base-medium and splenocytes were cultured to analyze the requirement of each cytokine in the induction of senescence-like Mφs. The generated Mφs were analyzed by SA-β-Gal assay. SA-β-Gal-positive cells in six different views were counted; the proportion of positive cells was calculated. The bars indicate the mean (±SD). Each dot represents the percentage of β-Galactosidase positive Mφs in each sample. *p<0.05, **p<0.01, Tukey-Kramer honest significant difference test. (C) The IL-6 gene was targeted in 8B cells by CRISPR/Cas9. Gray bar indicates 8B-mock; blue bar indicates 8B-IL-6-knockout (8B-IL-6-KO). (D) Cell proliferation activity was measured daily by the MTT assay. 8B-mock and 8B-IL-6-KO proliferated comparably in vitro. The symbols indicate the mean (±SD). Gray circles indicate 8B-mock; navy triangles indicate 8B-IL-6-KO. (E) Impact of 8B derived IL-6 on Mφs induction into a senescence-like state. Four-day culture supernatant of 8B-mock or 8B-IL-6-KO cells was used during splenic-F4/80+ cell culture. After 14 days, generated Mφs were observed by phase-contrast microscopy. Bars; 50 µm. (F) Gene expression of generated Mφs prepared in C was analyzed by quantitative reverse transcription PCR (RT-qPCR). The bars indicate the mean (±SD). Each dot represents the relative value normalized to Hprt of each sample. (G) One thousand 8B-mock or 8B-IL-6-KO cells were transplanted into NOD/SCID (8B-mock (gray) n=9, 8B-IL-6-KO (navy) n=11), or C57BL/6 mouse brain (8B-mock (gray) n=18, 8B-IL-6-KO (blue) n=19), and mouse survival was examined. **p<0.01, log-rank test.