Supplementary Figure 5: Correlation between high G-CSF expression and TIC features.

(a) 4T1 control cells and 4T1 cells expressing G-CSF shRNA were subjected to intracellular G-CSF FACS staining procedure. Overlay-graph demonstrates changes in G-CSF levels. The experiment was performed twice with similar results. Results of a representative experiment are shown. (b) Staining specificity control for pS6K antibody: PS6K levels were measured by intracellular FACS staining in 4T1 cells cultured in medium without serum (starved) or with 10% FBS. (c) Cells with highest G-CSF expression (top 5.3%, expression >2 times mean of total population) were compared to whole 4T1 cell population in the expression of CD24/CD29. Representative plots of gating strategy and identification of CD24^highCD29^high cells. (d). Same analysis as in (c), for P53N-C model. In (c) and (d): representative of three FACS staining experiments with 2–3 stained samples per experiment. (e) 4T1 cells were stained for G-CSF, CD24 and CD29. Expression of G-CSF in CD24^highCD29^high cells and in the total population was compared. (f) Same analysis as in (e), for P53N-C cells. (g,h). Correlation between G-CSF and Epcam⁺CD49f⁺ cells; analysis was done as described for CD24^highCD29^high cells in (e). In (h), EpCAM^highCD49f^med and EpCAM^medCD49f^high denote specific cell population in P53N-C model as described in Fig. 6c. (e–h) n = 3 independent experiments each with technical replicates. Mean values of all three experiments are used to generate the plot and calculate P values. (i) Under TIC enriched conditions 4T1 tumour cells produce increased levels of soluble factors to enhance MDSC differentiation from naïve bone marrow cells. Treatment of primary mouse bone marrow cells with conditioned medium (CM) from either 4T1 cells cultured in TIC-enriching condition (as 3D suspension mammospheres) or 2D conditions favouring differentiation. To eliminate effects of media-composition, all treatment conditions contained the same percentage of 2D and 3D media by supplementing with non-conditioned media as needed. αG-CSF: Five times ED₅₀ dose of G-CSF-neutralizing antibody. G-CSF: 40 ng ml⁻¹ recombinant human G-CSF. (j) Treatment of primary human bone marrow cells with CM from either MC1 cells cultured in 3D TIC-enriching condition or 2D conditions favouring differentiation. Left: FACS analysis showing MDSC-like cells in treated bone marrow. Right: quantification of MDSC-like cells under indicated conditions. Data are from two independent experiments. Error bars indicate s.e.m., and P values are calculated by two-tailed Student’s t tests. Statistics source data of Supplementary Fig. 5e–h are provided in Supplementary Table 4.