Figure 4A. Quantification of in vitro matrigel invasion assay of PANC-1 cells cultured for 48 h with conditioned media (CM) of NFs (P-NFs-CM), CAF low #44 and #110 and CAFs high #36 and 138. Histograms show the number of invading cells measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates, performed at least in duplicate each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. * P <0.05, **P<0.01, ***P<0.001. B. Quantification of in vitro matrigel invasion assay of PANC-1 cultured for 48 h with CM derived from control P-CAFs#36 (siCNT-P-CAF-CM#36) and hMENA/hMENA∆v6 silenced P-CAFs (sihMENA(t)-P-CAF-CM#36), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects PANC-1 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01. C. Quantification of in vitro matrigel invasion assay of H1975 cells cultured for 48 h with control media (culture medium) or conditioned media (CM) of L-CAF low #400 and CAFs high #189, as described above. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001. D. Quantification of in vitro matrigel invasion assay of H1975 cultured for 48 h with CM derived from control L-CAFs#484 (siCNT-L-CAF-CM#484) and hMENA/hMENA∆v6 silenced L-CAFs (sihMENA(t)-L-CAF-CM#484), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects H1975 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of six replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01. E. Representative immunoblot (top) and densitometry quantification (bottom) of hMENA∆v6 expression level in P-NFs grown in RPMI control medium (-) or PANC-1-CM for 24 h (n = 3). Data are represented as fold increase with respect to control medium ± SD (set as 1). Data were analyzed using two‐sided Student's t‐test. *P < 0.05. F. Representative images (top) and quantification (bottom) of collagen gel-contraction ability of CAFs (monoculture) or CAFs co-cultured with siCNT (co-siCNT) or sihMENA(t) PANC-1 cells (co-sihMENA(t)). Dashed white circles illustrate the margins of the gel area. Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P=0.005, followed by Bonferroni's multiple comparison test ANOVA: *P<0.05, **P<0.01. G. Representative immunoblot (top) and quantification (bottom) of hMENA∆v6 and α-SMA expression in P-CAFs #110 treated with DMEM medium (-) or with conditioned medium (CM) derived from siCNT (siCNT PANC-1) or sihMENA(t) PANC-1 cells ((sihMENA(t) PANC-1) (n = 3). Data are represented as fold increase with respect to DMEM (set as 1± SD). Data presented were analyzed using two‐sided Student's t‐test: *P<0.05.