Figure 8: Senescence-derived IL-6 is necessary to drive G-MDSC accumulation and tumour promotion.

(a) Senescence-associated β-galactosidase (SA-βgal) staining (blue) of shRFP-expressing MSFs treated with 10 μM TAM (Sen) or vehicle (Non-Sen) revealed ∼65% of cells were senescent following TAM treatment. Scale bar, 100 μm. n=3. (b) SA-βgal staining (blue) of shIL6#1-expressing MSFs treated with 10 μM TAM (Sen) or vehicle (Non-Sen) revealed ∼55% of shIL6 #1 cells were senescent following TAM treatment. Scale bar, 100 μm. n=3. (c) SA-βgal staining (blue) of shIL6#2-expressing MSFs treated with 10 μM TAM (Sen) or vehicle (Non-Sen) revealed ∼55% of shIL6 #2 cells were senescent following TAM treatment. Scale bar, 100 μm. n=3. (d) Quantification of SA-βgal-positive shRFP-, shIL6 #1-, and shIL6 #2-expressing cells shown in a–c. Data are mean %-positive cells+s.e.m. *P value <0.05, Student’s t-test. n=3. (e) qRT–PCR for IL-6 mRNA expression. Data are relative IL-6 mRNA in Sen/Non-Sen shIL6 lines compared with shRFP. n=3. Representative experiment. (f) Impact of senescent stromal-derived IL-6 on tumour growth. MSFs expressing IL-6-specific hairpins (shIL6 #1 or shIL6 #2) were co-injected with PDSC5 tumour cells. shRFP was used as a control. Data are mean fold growth of Sen co-injections over Non-Sen co-injections+s.e.m. Non-Sen injections are normalized to 1. *P value <0.05, Wald test. ns is not significant. n=5–10. (g) Flow cytometry of shRFP or shIL6 #1-expressing Non-Sen or Sen MSF isografts. Mice were injected with senescent or non-senescent MSFs that expressed a control shRFP or were depleted of IL-6 through the expression of the shIL6 #1 short hairpin. Data represents the mean number of G-MDSCs+s.e.m., per mouse. *P value <0.05, Student’s t-test. ns is not significant. n=3–4. (h) Flow cytometry of shIL6 expressing Non-Sen or Sen MSF-PDSC5 co-injections. Mice were injected with PDSC5 tumour cells and senescent or non-senescent MSFs depleted of IL-6 through the expression of shIL6 #1. Data represents the mean number of G-MDSCs+s.e.m., per mouse. ns is not significant, Student’s t-test. n=3–4. qRT–PCR, quantitative PCR with reverse transcription.