Fig. 2: Emergence and cell invasion rely on secreted proteins produced by emergent cells.

a Emergent cells were generated as described above and conditioned media (CM) collected after 24 h of serum starvation were supplemented with 10% FBS and applied to untreated parental LS174T cells. The proliferative capacity and survival were quantified by clonogenic tests (n = 6, Kolmogorov–Smirnov test, * = p < 0.05, ** = p < 0.01). b Conditioned media (CM) were collected after 24 h of serum starvation. LS74T cells were treated with sn38 for 4 days and emergence was induced after senescence induction for 7 days using either RPMI or conditioned medium from parental or emergent cells, both supplemented with 10% FBS. CM were added during the treatment and during emergence. Clones were then counted using crystal violet staining (n = 5, normalized to the emergence obtained from the CM of parental cells, Kolmogorov–Smirnov test, ** = p < 0.01). c Analysis of anoikis resistance and enhanced cell transformation using soft agar assays. Conditioned media obtained from parental or emergent cells were supplemented with 20% FBS and mixed with RPMI 0.7% low-melting-point (LMP) agarose to a final concentration of 10% FBS and 0.35% LMP agarose (n = 4). d Invasion assays were performed using Boyden inserts in which Matrigel was deposited at the bottom of the inserts. Conditioned media supplemented with 10% FBS were placed in the bottom chamber. After 72 h, invasive cells were stained with crystal violet (n = 5). e Migration assays were performed using Boyden inserts. Conditioned media obtained from parental or emergent cells supplemented with 3% FBS were placed at the well bottom. After 72 h, migrating cells were stained with crystal violet (n = 4). f CIS was induced in 4T1 cells following doxorubicin treatment (75 ng/ml, 4 days). Senescent cells mixed with untreated cells were injected in mice and tumor growth was monitored in the mammary fat pad of Balb/c mice and compared with controls (six mice were used for untreated 4T1 cells, seven for the mix of 4T1 cells and senescent population, and six for the senescent clones, two-way Anova with a Bonferroni’s multiple comparison test: ** = p < 0.01, p = 0.0018). g Following CIS induction, 4T1 cells were injected in the tail vein, alone or with senescent cells, and metastasis invasion was monitored after 31 days (Mann–Whitney test, * = p < 0.05)