Context-dependent inhibitory roles of RhoA in 3D invasive cell migration within the extracellular matrix

Abstract

Cell migration is fundamental to both physiological and pathological processes, including cancer progression. This study investigates the role of the small GTPase RHOA in invasive cell migration within diverse 3D extracellular matrix (ECM) environments using non-cancerous HEK293, pancreatic cancer PANC-1, and breast cancer MDA-MB-231 cells. Spheroid invasion assays showed that RHOA loss enhanced migration in HEK293 and PANC-1 cells cultured in Geltrex but not in type I collagen. In contrast, RHOA deletion had little effect on MDA-MB-231 migration in either ECM. Enhanced migration in RHOA-deficient HEK293 cells required protein phosphatase PTP1B and the small GTPases RAC and CDC42. Unexpectedly, while RHOA knockout increased 3D migration, it reduced pancreatic tumor progression in mice. These findings reveal that RHOA regulates cell invasion in a manner dependent on ECM composition and cellular context, highlighting its complex, context-specific roles and potential as a therapeutic target in cancer.

Keywords: 3D cell migration; CP: Cancer; CP: Cell biology; KPC; PDAC; RHOA; cancer; cancer invasion.

Figure 1.. RHOA KO promotes invasive cell migration of HEK293 spheroids into Geltrex

(A) Western blot analysis of WT and RHOA-KO HEK293 cells using antibodies to RHOA and GAPDH. (B) Spheroid invasion assays: (i) spheroids were generated using the hanging drop method and embedded in Geltrex or collagen I matrix. (ii) Single cells were seeded in Geltrex and allowed to form spheroids. (C) WT and RHOA-KO HEK293 (#2) spheroids were embedded in Geltrex and observed for 7 days using phase-contrast microscopy. Scale bar, 200 μm. (D) Quantification of inverse circularity (mean ± SD, n = 3 experiments, 20–40 spheroids per experiment). (E) Single WT and RHOA-KO HEK293 cells were seeded and monitored for 12 days. Scale bar, 50 μm. (F) Quantification of inverse circularity (mean ± SD, n = 3 experiments). (G) HEK293 spheroids were embedded in the collagen I matrix and observed. Scale bar, 200 μm. (H) Quantification of inverse circularity (mean ± SD, n = 3 experiments). (I) Analysis of 2D cell migration using a wound-healing assay. Representative phase-contrast images of WT and RHOA-KO HEK293 cells at 0, 1, 2, and 3 days. Scale bar, 400 μm. (J) Wound closure quantification (relative width over initial width, mean ± SD, n = 3 experiments). Statistical analysis: one-way ANOVA with post hoc Tukey (D, F, H, and J): *p < 0.05 and ***p < 0.001.

Figure 2.. RHOA KO does not affect invasive cell migration of MDA-MB-231 spheroids

(A) Western blot analysis of WT and RHOA-KO MDA-MB-231 cells using antibodies to RHOA and GAPDH. (B) WT and RHOA-KO MDA-MB-231 (#1) spheroids were embedded in Geltrex and observed using phase-contrast microscopy. Scale bar, 200 μm. (C) Quantification of the radius of the migrating front circle (mean ± SD, n = 3 experiments, ~30–70 spheroids per experiment). (D) Single WT and RHOA-KO MDA-MB-231 cells were seeded and monitored for 12 days. Scale bar, 50 μm. (E) Quantification of the radius of the migrating front circle (mean ± SD, n = 3 experiments). (F) MDA-MB-231 spheroids were embedded in the collagen I matrix and observed. Scale bar, 200 μm. (G) Quantification of the radius of the migrating front circle (mean ± SD, n = 3 experiments). (H) Analysis of 2D cell migration using a wound-healing assay. Representative phase-contrast images of WT and RHOA-KO MD-MBA-231 cells at 0, 16, 24, and 40 h. Scale bar, 400 μm. (I) Wound closure quantification (relative width over initial width, mean ± SD, n = 3 experiments). Statistical analysis: one-way ANOVA with post hoc Tukey (C, E, G, and H).

Figure 3.. RHOA depletion increases invasive cell migration of PANC-1 spheroids into Geltrex

(A) Western blot analysis of WT and RHOA-depleted PANC-1 cells using antibodies to RHOA and GAPDH. (B) PANC-1 spheroids were generated using the hanging drop method, embedded in Geltrex, and monitored for 7 days using phase-contrast microscopy. Scale bar, 200 μm. (C and D) The migration area (C) was quantified by subtracting the spheroid area (D) (indicated by the red line) from the total area (indicated by the yellow line) (mean ± SD, n = 3 experiments, ~20–50 spheroids per experiment). (E) Analysis of 2D cell migration using a wound-healing assay. Representative phase-contrast images of WT and RHOA-knockdown PANC-1 cells at 0, 1, 2, and 3 days. Scale bar, 400 μm. (F) Wound closure quantification (relative width over initial width, mean ± SD, n = 3 experiments). Statistical analysis: one-way ANOVA with post hoc Tukey (C, D, and F): ****p < 0.0001.

Figure 4.. RHOA loss promotes filopodia formation in RHOA-HEK293 cells

(A) WT and RHOA-KO HEK293 cells were cultured in Geltrex for 24 h and then fixed and stained with Alexa 568-phalloidin (green). DAPI staining was included for 3D cultures. Scale bar, 20 μm. (B) Filopodia density (mean ± SD, n = 3 experiments). (C) Filopodia length (mean ± SD, n = 3 experiments). Statistical analysis: two-tailed Student’s t test (B, C, and E): *p < 0.05 and **p < 0.01.

Figure 5.. PTP1B is critical for the increased invasive migration of RHOA-KO HEK293 cells in Geltrex

(A) Single WT and RHOA-KO HEK293 cells were seeded in Geltrex and monitored for 12 days in the presence of 30 μM PTP1B-IN-4. The culture medium containing PTP1B-IN-4 was replaced every 4 days. Scale bar, 50 μm. (B) Quantification of inverse circularity (mean ± SD, n = 3 experiments). (C) Wound-healing assay was performed in the presence of 30 μM PTP1B-IN-4 using WT and RHOA-KO HEK293 cells. Scale bar, 400 μm. (D) Quantification of wound closure (relative wound width over initial width, mean ± SD, n = 3 experiments). Statistical analysis: one-way ANOVA with post hoc Tukey (B and C): *p < 0.05.

Figure 6.. Increased invasive migration of RHOA-KO HEK293 cells requires RAC1 and CDC42 in Geltrex

(A–D) Single WT and RHOA-KO HEK293 cells were seeded in Geltrex and cultured for 4 days in the presence of 10 μM EHT1864 (A), NSC-23766 (B), ML141 (C), or MLS-57351 (D). Scale bar, 50 μm. Quantification of inverse circularity at day 7 is shown (mean ± SD, n = 3 experiments). (E) RHOA, RAC1, and CDC42 activity in WT and RHOA-KO HEK293 cells in 2D and 3D spheroid cultures (mean ± SD, n = 3–4 experiments). (F) Western blot analysis of WT and RHOA-KO HEK293 spheroids using the indicated antibodies. (G) Quantification of band intensity (mean ± SD, n = 3 experiments). Statistical analysis: one-way ANOVA with post hoc Tukey (A–E) and two-tailed Student’s t test (G): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 7.. RHOA KO delays PDAC progression and alters tumor morphology

(A) Histological analysis (H&E and Masson’s trichrome staining). Scale bar, 200 μm. The enlarged boxed regions highlight details. Scale bar, 100 μm. (B) PDAC progression stages (n = 7–10 mice for each genotype). (C) Immunohistochemistry of pancreas stained for Ki67 and α-SMA. Scale bars, 20 μm for Ki67 and 100 μm α-SMA. (D) Quantification of Ki67-positive nuclei and α-SMA-positive area (mean ± SD, n = 4 mice for each genotype). (E) Representative pancreas images. Scale bar, 1 cm. (F) Pancreas weight quantification (mean ± SD, n = 6 WT, 7 KPC, 9 KPC::RHOA-KO mice). (G) Survival curve of KPC and KPC::RHOA-KO mice (n = 10 mice for each genotype). (H) H&E-stained pancreas sections of KPC and KPC::RHOA-KO mice at 12 weeks post-tamoxifen injection. Scale bar, 1 cm. Tumor boundaries are outlined; boxed regions are enlarged. (I) Quantification of inverse circularity of tumors (mean ± SD, n = 20 sections from 6 KPC mice and 50 sections from 10 KPC::RHOA-KO mice). Statistical analysis: one-way ANOVA with post hoc Tukey (D and F), log rank Mantel-Cox test (G), and two-tailed Student’s t test (I): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.