The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/β-Catenin Signaling

Tumor cells progressively remodel cytoskeletal structures and reduce cellular stiffness during tumor progression, implicating the correlation between cell mechanics and malignancy. However, the roles of tumor cell cytoskeleton and the mechanics in tumor progression remain incompletely understood. We report that softening/stiffening tumor cells by targeting actomyosin promotes/suppresses self-renewal in vitro and tumorigenic potential in vivo. Weakening/strengthening actin cytoskeleton impairs/reinforces the interaction between adenomatous polyposis coli (APC) and β-catenin, which facilitates β-catenin nuclear/cytoplasmic localization. Nuclear β-catenin binds to the promoter of Oct4, which enhances its transcription that is crucial in sustaining self-renewal and malignancy. These results demonstrate that the mechanics of tumor cells dictate self-renewal through cytoskeleton–APC–Wnt/β-catenin–Oct4 signaling, which are correlated with tumor differentiation and patient survival. This study unveils an uncovered regulatory role of cell mechanics in self-renewal and malignancy, and identifies tumor cell mechanics as a hallmark not only for cancer diagnosis but also for mechanotargeting.

sphere formation assay at passage 4 (C) compared to EpCAM-cells.Fluorescence-activated cell sorting (FACS) was utilized to sort EpCAM+/-subpopulations from Huh-7 cells.The selfrenewal of the sorted tumor cells was tested in soft fibrin and soft agar.n=3.EpCAM+ cells exhibit lower F-actin (D, E) and cellular stiffness (F) than EpCAM-cells.F-actin was examined by phalloidin staining and cell stiffness was measured by atomic force microscopy.n>60 cells in (E) and n>90 cells in (F).(G, H) Fibrin-selected CSCs up-regulate self-renewal genes.HCC CSCs were obtained by culturing Huh-7 cells in soft fibrin gels for 7 days.The expressions of the indicated self-renewal genes were measured by both qRT-PCR (H) and western blotting (G).The results in (G) were representative of two independent experiments.n=3 in (H).Fibrinselected CSCs show lower F-actin (I, J) and cellular stiffness (K) than control cells.n>70 cells in (J).n>110 cells in (K).(L, M) EpCAM+ cells and fibrin-selected CSCs generate higher contractility than EpCAM+ cells and control cells.Cellular contractility was represented by traction force measured by traction force microscopy.n>24 cells for each condition.Scale bar in (D and I): 50 µm.Mean ± SEM.One-way ANOVA and student t-test were used for the statistical analysis in (A, B, E, and F) and (H and J-M), respectively.*, p<0.05; **, p<0.01; ***, p<0.001.ns: no significance.

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Figure S1 Tumor cell mechanics are correlated with self-renewal in HCC.EpCAM+ HCC cells generate larger tumor spheroids in soft fibrin (A) and more colonies in soft agar (B) and

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Figure S3 Modulating cell mechanics has no significant influence on cell morphology, focal adhesion, microtubule, and intermediate filament while affecting cellular contractility.(A-C) Softening cells has no effect on cell morphology.Huh-7 cells were transfected with the siRNAs of MLCK and mDia1.The spreading area and aspect ratio of these cells were measured.n>100 cells in (B, C). (D-F) Softening cells has no effect on focal adhesion, microtubule, and intermediate filament.The expressions of vinculin, microtubule, and vimentin were measured in the treated cells by immunofluorescence staining.n>60 cells in (D); n>100 in (E, F). (G) Softening cells reduces cellular contractile forces.Cellular contractility was measured by traction force microscopy.n>18 cells for each condition.(H-J) Stiffening cells has no effect on cell morphology.Huh-7 cells were transfected with CA-MLCK

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Figure S4 Softening cells enhances tumor cell self-renewal.(A, B) Softening tumor cells up-regulates self-renewal markers.Huh-7 cells were transfected with the siRNAs of MLCK and mDia1.The expressions of CD133 (A), Bmi1, and other self-renewal genes (B) were measured by immunofluorescence staining, western blotting, and qRT-PCR, respectively.n>80 cells in (A) and n=3 in (B).Softening cells promotes the formation and growth of tumor spheroids and the CSC fraction in Hep3B (C-E), HepG2 (F-H), and MHCC97L (I, J).Various HCC cells were transfected with the siRNAs of MLCK and mDia1 and their self-renewal was tested in soft fibrin (C, F, and I) and soft agar (D, G, and J).The fractions of EpCAM+ (E) and CD90+ (H) cells were measured by flow cytometry.n=3.(K-N) Pharmacologically softening

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Figure S6 Considerable cell softening suppresses self-renewal.(A, B) The knockdown efficiency of the siRNAs of MLCK and mDia1.n=3.(C, D) High doses of siRNAs further reduce F-actin and cellular stiffness.Huh-7 cells were transfected with 10 nM and 100 nM of MLCK and mDia1 siRNAs, respectively.F-actin and cell stiffness were measured by phalloidin staining and atomic force microscopy, respectively.n>100 in (C); n>70 in (D).Scale bar: 50 µm.(E, F) Moderate/considerable cell softening promotes/suppresses self-renewal.The self-renewal of the treated cells in (C) was tested in soft agar, SFA (E), and soft fibrin (F).n=3.(G) High doses of inhibitors further reduce cellular stiffness.Huh-7 cells were treated with 0.1 and 1 µM Cytochalasin D (CytoD), 2 and 20 µM Y27632, and 6 and 50 µM Blebbistatin (Bleb), respectively.Cell stiffness was measured by atomic force microscopy.n>70.Considerable cell softening suppresses the formation (H) and growth (I) of tumor spheroids.n=3.(J-L) Considerable softening does not affect cell viability but suppresses proliferation.The viability

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Figure S7 Disrupting microtubule and vimentin suppresses self-renewal.Pharmacologic treatment reduces microtubule (A) and vimentin (E).Huh-7 cells were treated with Nocodazole (1 and 6 µM) and Withaferin A (WFA; 1, 2, and 5 µM), respectively.The expressions of αtubulin and vimentin were measured by immunofluorescence staining.n>100 cells.(B, C, F, G) Disrupting microtubule and vimentin suppresses the formation and growth of tumor spheroids.The self-renewal of the treated cells in (A, E) was tested in soft fibrin (B, F) and soft agar (C, G). n=3.(D, H) Disrupting microtubule and vimentin has minimal/suppressive effect on cell proliferation.The proliferation of the treated cells was measured at the indicated time points by MTS assay.n=3.One-way ANOVA was used for the statistical analysis.*, p<0.05; **, p<0.01; ***, p<0.001.

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Figure S8 Softening/stiffening cells promotes/suppresses self-renewal in multiple types of cancer.Moderately softening tumor cells enhances self-renewal in breast (MCF-7; A), cervical (HeLa; B), colon (CT26; C), and lung (A549; D) cancer.Tumor cells were transfected with the siRNAs of MLCK and mDia1.The self-renewal of these cells was tested in soft fibrin and soft agar, respectively.Stiffening tumor cells suppresses self-renewal in breast (MCF-7; E), cervical (HeLa; F), colon (CT26; G), and lung (A549; H) cancer.Tumor cells were transfected with CA-MLCK and CA-ROCK plasmids.The self-renewal of these cells was tested in soft fibrin and soft agar, respectively.n=3.One-way ANOVA was used for the statistical analysis.*, p<0.05; **, p<0.01; ***, p<0.001.

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Figure S9 Stiffening cells suppresses self-renewal in multiple types of HCC cells.(A, B) Stiffening cells down-regulates multiple self-renewal genes.Huh-7 were transfected with CA-MLCK and CA-ROCK plasmids.The expressions of the indicated self-renewal genes were measured by western blotting, qRT-PCR (A), and immunofluorescence staining (B).n=3 in (A); n>80 in (B).(C-E) Stiffening cells suppresses self-renewal in multiple HCC cells.MHCC97L (C) and PLC/PRF/5 cells (D, E) were transfected with CA-MLCK and CA-ROCK plasmids.Their self-renewal was tested in soft fibrin and soft agar.n=3.(F) Stiffening CSCs reduces the growth of tumor spheroids in soft fibrin.n=3.(G) Pharmacologic treatment increases cellular stiffness.Huh-7 cells were treated with 20 nM Jasplakinolide (Jas) or 1 nM Narciclasine (Narci).The stiffness of these treated cells was measured by atomic force microscopy.n>110 cells.(H-L) Pharmacologically stiffening HCC cells and CSCs suppresses self-renewal.The self-renewal of the treated cells was tested in soft fibrin and agar (H, I, L).The fraction of EpCAM+ cells was measured by flow cytometry (J).The expressions of

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Figure S10 Dynamically modulating tumor cell stiffness regulates their self-renewal in a reversible manner.Dynamic alterations of tumor cell stiffness by activating CA-MLCK and CA-ROCK plasmids influence the growth of tumor spheroids of HCC cells (A, B) and CSCs (C, D) in soft fibrin.Huh-7 cells and CSCs were transfected with CA-MLCK and CA-ROCK plasmids and then cultured in soft fibrin with or without the induction of doxycycline (Doxy).-/-Doxy: without Doxy during the entire period; -/+Doxy: without Doxy during the first 6 days while with Doxy during the last 6 days; +/-Doxy: with Doxy during the first 6 days while without Doxy during the last 6 days; +/+Doxy: with Doxy during the entire period.n=3.Oneway ANOVA was used for the statistical analysis.*, p<0.05; **, p<0.01; ***, p<0.001.

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Figure S11 Cell mechanics alter transcriptomic profiles of HCC cells.(A) Heatmap of gene expression of Huh-7 cells in control, si-MLCK, and si-mDia1 groups from RNA-seq analysis.n=3.(B) Principal component analysis (PCA) showing distinct separation of control, si-MLCK, and si-mDia1 groups.(C, D) The Metascape ontology analysis of upregulated genes following knockdown of mDia1 and MLCK in Huh-7 cells.The enriched ontologies related to WNT/βcatenin signaling and stemness were shown.(E, F) GSEA of CHIANG_LIVER_CANCER_SUBCLASS_CTNNB1_UP gene set, as defined by top 200

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Figure S13 The alteration of cell stiffness is involved in CSC differentiation.(A) HCC CSCs increase cell stiffness after culture on glass.Fibrin-selected CSCs were cultured on glass for 7 days in the presence of Y-27632 or DMSO.The stiffness of these cells was measured by atomic force microscopy.Control: control Huh-7 cells.n>120 cells.(B-E) CSCs become differentiated after culture on glass, which is suppressed by cell softening.The fraction of EpCAM+ cells was measured in the treated cells (B).The self-renewal was tested in soft fibrin (C) and agar (D).The expression of CD133 was analyzed using immunofluorescence staining

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Figure S14 Cell mechanics regulate the interconversion between CSCs and non-CSCs in a Wnt-dependent manner.(A-C) Cell softening facilitates the de-differentiation of EpCAMcells into EpCAM+ cells.EpCAM-Huh-7 cells were treated with 0.1 µM Cytochalasin D (CytoD), 2 µM Y27632, and 6 µM Blebbistatin (Bleb).The fraction of EpCAM+ cells was measured by flow cytometry (A) and the self-renewal of these treated cells was tested in soft fibrin (B) and agar (C).n=3.(D-F) Cell stiffening facilitates the differentiation of EpCAM+ cells into EpCAM-cells.EpCAM+ cells were treated with 20 nM Jasplakinolide (Jas) and 1 nM Narciclasine (Narci).The fraction of EpCAM+ cells was measured by flow cytometry (D) and the self-renewal of these treated cells was tested in soft fibrin (E) and agar (F).n=3.(G-I) Cell softening mediated de-differentiation of EpCAM-cells into EpCAM+ cells depends on Wnt signaling.EpCAM-cells were treated with 0.1 µM CytoD, 2 µM Y27632, and 6 µM Bleb in the presence or absence of IWR.The self-renewal of these treated cells was tested in soft

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Figure S15 Cell mechanics regulate tumor cell self-renewal through APC/Wnt/β-catenin signaling.(A-D) Cell mechanics affect the expression of APC.Huh-7 cells were transfected with the siRNAs of MLCK and mDia1 or CA-MLCK and CA-ROCK plasmids.The expression of APC was measured at both mRNA (A) and protein levels (B).The fraction of APC+ cells was analyzed by flow cytometry (C, D). n=3 in (A, C, D); n>50 cells in (B).(E) CSCs express

Figure S16
Figure S16 The influence of cell mechanics on the cytoskeleton and self-renewal.(A, B) Softening/stiffening cells decreases/increases the co-localization between APC and F-actin.n>15 cells.(C, D) Cell mechanics influence the expression of α-catenin.n=3.(E, F) Knockdown and overexpression of Oct4.Huh-7 cells were transfected with Oct4 siRNA or