Journal: eBioMedicine
Article Title: Migrasome-mediated clearance of excess PLK4 defines a targetable vulnerability
doi: 10.1016/j.ebiom.2026.106237
Figure Lengend Snippet: Targeting TSPAN6 suppresses tumour growth and metastasis. (A) Real-time cell proliferation assay of PLK4−OE cells treated with scramble or shTSPAN6 RNA. Data are presented as mean values ± SD. (B) Cystal violet staining of shTSPAN6 or control PLK4−OE cells after 14 days of culture. Colony number of each group was quantified on the right. Data are presented as the mean ± SD from three independent repeats. p values were calculated using unpaired two-tailed Student's t-tests, with p < 0.05 considered statistically significant. (C) Fluorescence staining for active apoptosis in shTSPAN6 or control PLK4−OE cells detected by GreenNuc Caspase-3 Assay Kit. The green fluorescence in nuclei indicate active apoptosis. Scale bar, 20 μm. (D) Western blot analysis of cleaved caspase3 in PLK4−OE cells treated with scramble or shTSPAN6 RNA. β-actin was used as the loading control. (E) Western blot analysis of PLK4 protein levels in primary tumour cells derived from 18 patients with triple-negative breast cancer (TNBC). β-Actin served as the loading control (left). Relative PLK4 protein levels were quantified as the ratio of sample to NC (sample/NC). Cases with sample/NC > 1 were classified as PLK4 high (red dots), whereas cases with sample/NC < 1 were classified as PLK4 low (blue dots) (right). (F) Growth curve of tumours in PDX-models treated with scramble or shTSPAN6 RNA. Tumour volumes are presented as mean values ± SD (n = 30 mice per group). p values were calculated by two-way ANOVA. p < 0.05 was considered statistically significant. (G, H) Immunohistochemical staining of Ki67 and cleaved-caspase3 in tumours from PDX-mice treated with scramble or shTSPAN6 RNA. Scale bar, 100 μm. (I) Immunofluorescent staining for PLK4, TSPAN6, and α-tubulin in frozen tumour sections from PLK4 high tumour mice treated with scramble or shTSPAN6 RNA. White arrowheads indicate PLK4 particles colocalized with TSPAN6 in the extracellular space. Yellow arrowheads indicate bipolar spindles in the control group and multipolar spindles in the shTSPAN6 RNA-treated group. Scale bar, 20 μm for each main image and 2 μm for the zoom in section. Regions marked by the arrowheads are shown at higher magnification in the right three columns. (J) Quantification of PLK4-containing particles in the extracellular space of frozen tumour sections from PLK4 high tumour mice treated with scramble or shTSPAN6 RNA. 20 fields of view from tumour sections of three mice per group were analysed. Data are presented as mean ± SD. p values were calculated using unpaired two-tailed Student's t-tests. p < 0.05 was considered statistically significant. (K) Proportion of bipolar spindles in mitotic tumour cells in frozen tumour sections from PLK4 high tumour mice treated with scramble or shTSPAN6 RNA. (L) Western blot analysis of intracellular PLK4 protein levels in PLK4 high tumours derived from PDX-mice treated with scramble or shTSPAN6 RNA. β-Actin was used as the loading control. Quantification is presented as mean ± SD from three independent biological replicates. p values were calculated using unpaired two-tailed Student's t-tests, with p < 0.05 considered statistically significant. (M) Liver and lung tissues were harvested from PDX-mice with or without detectable metastatic lesions. Arrowheads indicate representative metastatic nodules within the organs. (N) Metastatic incidence in the liver and lung tissues of PDX-mice treated with scramble or shTSPAN6 RNA. The number of mice analysed in each group is indicated above the corresponding bar. (O) Metastatic regions in liver and lung tissues visualised by H&E staining. Representative metastatic foci are indicated by arrowheads, with corresponding higher-magnification images shown on the right. Scale bar, 100 μm for each main image and 20 μm for the zoom in section. (P) Quantification of metastatic nodules in the lungs and livers of PDX-mice treated with scramble or shTSPAN6 RNA. Each dot represents an individual mouse bearing metastatic nodules in the indicated organ.
Article Snippet: Primary antibodies were obtained as follows: anti-PLK4 (Cat# MABC544, RRID: AB_2893410 ) and anti-Centrin (Cat# 04–1624, RRID: AB_10563501 ) from Millipore; anti-LAMP1 (Cat# ab25630, RRID: AB_470708 ), LC3B (Cat# ab192890, RRID: AB_2827794 ), Proteasome 20S alpha + beta (Cat# ab22673, RRID: AB_2268907 ), and LAMP2A (Cat# ab18528, RRID: AB_775981 ) from Abcam; anti-ERp72 (Cat# 66365-1-Ig, RRID: AB_2881745 ), GM130 (Cat# 11308-1-AP, RRID: AB_2115327 ), LMAN2 (Cat# 11496-1-AP, RRID: AB_3085375 ), TMED10 (Cat# 15199-1-AP, RRID: AB_2204321 ), ITGB1 (Cat# 12594-1-AP, RRID: AB_2130085 ), and TSPAN6 (Cat# 12293-1-AP, RRID: AB_2213446 ) from Proteintech; anti-cleaved caspase-3 (Cat# 9664, RRID: AB_2070042 ), PDI (Cat# 2446, RRID: AB_2298935 ), and CYCS (Cat# 4272, RRID: AB_2090454 ) from Cell Signalling Technology; anti-HSC70 (Cat# NB120-2788, RRID: AB_2120309 ) and Ki67 (Cat# NB500-170, RRID: AB_10001977 ) from Novus; anti-PIGK (Cat# PA5-28337, RRID: AB_2545813 ), EOGT (Cat# MA5-53414, RRID: AB_3247885 ), TSPAN4 (Cat# PA5-69344, RRID: AB_2688603 ) and Calnexin (Cat# PA5-34754, RRID: AB_2552106 ) from Thermo Fisher Scientific.
Techniques: Proliferation Assay, Staining, Control, Two Tailed Test, Fluorescence, Caspase-3 Assay, Western Blot, Derivative Assay, Immunohistochemical staining
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