Fig 2: Rnd3 silencing promotes entosis in hepatocellular carcinoma cells.A The inhibition of Rnd3 expression was performed in Hep3B (upper panel) and in Huh7 (lower panel) cells using either a shRNA targeting Rnd3 (ShRnd3) inducible with doxycycline treatment and compared to a control shRNA (ShCtrl) or three independent siRNA targeting Rnd3 SiRnd3 #1, SiRnd3 #2, SiRnd3 #3 and SiCtrl as control. Rnd3 knock-down was assessed by Western blot, β-actin is the loading control. Graphs show the quantification of entotic cells upon Rnd3 KD. Error bars: SD of three or more independent experiments. Significance was determined with the Mann–Whitney U-test. B, C Representative of a single (B) or a double (C) event of entosis after silencing of Rnd3 expression in Hep3B cells with siRnd3 (B, upper panel) or with shRnd3 (B, lower panel; and C). Cells were labeled with DAPI, phalloidin, and beta-catenin antibody to visualize the nucleus, actin, and membrane, respectively. Scale bar, 15 µm. D Entosis was visualized in fixed tumor tissues from mice subcutaneously inoculated with Hep3B-shCtrl (n = 3) or Hep3B-shRnd3 (n = 5) cells. Tumor sections were stained with hematoxylin-eosin (HE) and the percentage of entotic cells was quantified. Scale bar, 50 µm. Error bars: SD of three or more independent samples. Significance was determined with the Mann–Whitney U-test.

Fig 3: Characterization of entosis mediated by the loss of Rnd3 expression.A Dynamics of entosis upon Rnd3 silencing. Hep3B cell lines were transduced with H2B-GFP (yellow) and LifeAct-mRuby (cyan) to mark the nucleus and the actin, respectively. Cells were transfected with siRNA targeting Rnd3 and spinning-disk microscopy analysis was done over 48 h. The gallery corresponds to Video 1. Time is in hours. Three stages were defined: (i) cell contact, (ii) internalization, arrows show the internalization of the inner binuclear cell 21 h after contact between cells and (iii) degradation of the inner cell; the inner cell degradation starts about 2 h after internalization and the cell degradation takes almost 10 h to be completed. Scale bar, 28 µm. B Analysis of entosis by correlative light-scanning electron microscopy. Entotic cells characterized by the nucleus (H2B-GFP, green) deformation and high concentration of actin (LifeAct-mRuby, red) in the inner cell were chosen using immunofluorescence microscopy (scale bar, 100 µm) and then the same event was analyzed by scanning electron microscopy. Entotic cells were taken with two magnifications 1000x (scale bar, 5 µm) and 2000x (scale bar, 10 µm). C Two populations of Hep3B cells were mixed, one population transduced with H2B-GFP, and another one transduced with H2B-RFP and transfected with siRNA targeting Rnd3. 48 h after mixing, the quantification of entotic cells was performed by evaluating the percentage of different combinations, Green in Green (wild-type in wild-type cells), Green in Red (Wild-type in siRnd3-transfected cells), Red in Green (siRnd3-transfected in wild-type) and Red in Red (siRnd3-transfected in siRnd3-transfected cells). Error bars: SD of three or more independent experiments. Significance was determined with the Mann–Whitney U-test. Examples of events counted after mixing of Rnd3-silencing RFP cells and wild-type GFP cells; G in G = Green in Green, G in R = Green in Red, R in G = Red in Green, R in R = Red in Red. Scale bar, 15 µm.

Fig 4: Entosis induced by Rnd3 silencing is dependent on Rho/ROCK pathway and occurs independently of E-cadherin expression.A Hep3B cells were transfected with siRNA targeting Rnd3 (siRnd3 #1) in the presence or absence of siRNA targeting RhoA (siRhoA) (left panel), or after treatment or not with Y-27632 (right panel) and the percentage of entosis was evaluated. B Hep3B or Huh7 cell lines were treated with siRNA targeting p190RhoGAP-A (siP190A #1 and siP190A #2). The Western blots show the inhibition of p190A and the graphs indicate the percentage of entotic cells. C Hep3B or Huh7 cells were transfected with siRNA targeting Rnd3 in the presence or absence of siRNA targeting E-cadherin (siEcad #1 or siEcad #2). The inhibition of Rnd3 and E-cadherin was confirmed by Western blot and the percentage of entotic cells was evaluated. Error bars: SD of three or more independent experiments. Significance was determined with the Mann–Whitney U-test.

Fig 5: Working model depicting RTK and Rho GTPase signalling crosstalk and its impact on GBM cell invasion. (Left) RTKs are activated through diverse molecular mechanisms, sustaining high Rnd3 expression levels in glioma cells. Rnd3 antagonizes the RhoA/ROCK signalling pathway, leading to low basal RhoA activity and dephosphorylation of ROCK downstream effectors, such as cofilin. This Rho GTPase balance modulates actin cytoskeleton dynamics favouring inhibition of stress fibre formation and focal adhesion maturation, thus leading to a more fluid and highly motile phenotype. (Right) Inhibition of different RTKs with a panel of small-molecule tyrosine kinase inhibitors (1) reduces Rnd3 expression, which, in turn, allows for increased RhoA/ROCK signalling. This switch in Rho GTPase activity orchestrates actin cytoskeleton remodelling, characterised by formation of robust and thick stress fibres, allowed by increased cofilin phosphorylation and focal adhesion maturation, resulting in a more rigid and less motile phenotype. This phenotype is also recapitulated upon Rnd3 shRNA-mediated depletion (2).