Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: Wnt5a is overexpressed in prostate cancer and associated with VM formation mediated by cancer cell stemness and EMT. (A) The mRNA levels of Wnt5a were measured in a normal prostate epithelial cell line (RWPE-1) and prostate cancer cell lines (PC3, LNCaP, DU145) using quantitative polymerase chain reaction (qPCR), n = 3, * P ๤0.05 v.s. RWPE-1. (B, C) Western blot analysis was conducted to detect Wnt5a expression in the indicated cell lines, * P ๤0.05 v.s. RWPE-1, n = 3. (D-E) Immunohistochemical staining of WNT5A in tissues of BPH and PCa. D is a representative graph and E is a statistical graph, Original magnification, × 200; scale bar, 20 μm. * P ๤0.05 v.s. BPH, n = 50. (F) Representative images of CD31/PAS double staining and immunohistochemistry (IHC) staining were obtained from paraffin-embedded human prostate cancer specimens. The VM structure, indicated by the red arrow, exhibited a positive reaction to PAS staining and lacked CD31 staining. Additionally, the VM structure contained red blood cells, as observed in the magnified inset. On the other hand, the endothelium-dependent vessel, denoted by the black arrow, displayed positive staining for both CD31 and PAS, as observed in the magnified inset. Furthermore, the expression of Wnt5a was found to be higher in cases positive for VM (left) compared to cases negative for VM (right). Conversely, the marker for cellular epithelial characteristic, E-cadherin, was downregulated in cases positive for VM (left) compared to cases negative for VM (right). The expression levels of EMT-related protein (Vimentin) and CSC-associated protein (CD133) were found to be significantly higher in the VM positive cases (left) compared to the VM negative cases (right). Original magnification, × 200; scale bar, 20 μm, for insets, 10 μm.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemical staining, Staining, Double Staining, Immunohistochemistry, Marker

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: The correlation among VM, Wnt5a expression and EMT/CSCs-related proteins expressions in PCa tissues.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Expressing

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: Wnt5a functions as a regulator of VM formation mediated by EMT and stemness in PCa cell lines. (A) To investigate the role of Wnt5a in VM formation, DU145 cells were stably transfected with negative control shRNA lentivirus (stable-sh-Ctl) or shRNA lentivirus targeting Wnt5a (stable-shWnt5a). Subsequently, stable-shWnt5a cells were transiently transfected with either an empty vector plasmid (stable-shWnt5a+Vector) or a Wnt5a overexpression plasmid (stable-shWnt5a+Wnt5a-OV). The expression of Wnt5a in various cell groups was assessed using Western blot analysis. (B) The statistical results of gray values in (A) were presented as histogram. (C) LNCaP cells were transfected with either empty lentivirus (referred to as stable-Ctl) or lentivirus overexpressing Wnt5a (referred to as stable-Wnt5a). Subsequently, stable-Wnt5a cells were transiently transfected with negative control siRNA (referred to as stable-Wnt5a+NC) or siRNA specifically targeting Wnt5a (referred to as stable-Wnt5a+ si-Wnt5a). Western blot was applied to measure the expression of Wnt5a. (D) The statistic results of Western blot bands in (C). (E) The mRNA level of EMT-related genes (E-Cadherin, Vimentin), cell stemness-related genes (CD133, Nanog, Sox2, Oct4) and VM-related genes (VE-cadherin, MMP9) in DU145 cells from various groups were assessed using qPCR. (F, G) The expression levels of EMT-related proteins (E-Cadherin, Vimentin), cell stemness-related proteins (CD133, Nanog, Sox2, Oct4) and VM-related proteins (VE-cadherin, MMP9) in DU145 cells from different groups were determined through Western blot analysis. The grayscale statistical map of (F) was denoted as (G). (H-J) The relative expression levels of EMT-related genes, cell stemness-related genes and VM-related genes in LNCaP of different groups as indicated were measured using qPCR (H) and Western blot (I, J), (J) is the grayscale statistical map of (I). (K, L) Flow cytometric quantification was employed to determine the CD133-positive population in DU145 across different groups. The figures (K) and statistical chart (L) presented on the panel illustrate the CD133-positive population in DU145 across different groups. (M, N) Representative flow plots (M) and statistical chart (N) of CD133-positive in LNCaP of different groups are shown. (O, P) Representative images (O) and statistical chart (P) depict the tube formation in DU145. Original magnification, × 200; scale bar, 200 μm. (Q, R) Representative images (Q) and statistical chart (R) illustrate the tube formation in LNCaP. Original magnification, × 200; scale bar, 200 μm. * P <0.05 v.s. as indicated, n = 3.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Stable Transfection, Transfection, Negative Control, shRNA, Plasmid Preparation, Over Expression, Expressing, Western Blot

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: Wnt5a facilitates VM formation mediated by EMT and cellular stemness in vivo. (A) The live imaging presented depicts the growth of tumors in mice at day 7, 14, and 21 subsequent to orthotopic-injection of DU145 cells. The experimental groups include stable-sh-Ctl, which represents DU145 cells with stable transfection of negative control shRNA lentivirus, and stable-shWnt5a, which represents DU145 cells with stable transfection of shRNA lentivirus targeting Wnt5a. (B) The statistical diagram of (A) is shown, * P <0.05 v.s. stable-sh-Ctl, n = 6. (C) The image corresponds to the tumor samples obtained on day 21. (D) Statistic graph of tumor size of (C), * P <0.05 v.s. stable-sh-Ctl, n = 6. (E) Live imaging map of tumor in mice at day 7, 14 and 21 after LNCaP as indicated orthotopic-injection. stable-Ctl, LNCaP with stable transfection of empty-vector lentivirus; stable-Wnt5a, LNCaP with stable transfection of Wnt5a overexpression lentivirus. (F) Shown is the statistical diagram of average chemiluminescence intensity in (E), * P <0.05 v.s. stable-Ctl, n = 6. (G) The image of the tumor samples at day 21. (H) Statistic graph of tumor size of (G), * P <0.05 v.s. stable-Ctl, n = 6. (I) Shown is CD31/PAS double staining for identification of VM structure and IHC staining for Wnt5a as well as VM-associated factors (VE-cadherin, MMP9) in tumor sections obtained from DU145 orthotopic-injected mouse. Original magnification, × 100; scale bar, 100 μm. (J-K) The statistical chart of (I), * P <0.05 v.s. stable-sh-Ctl, n = 6. (L) Images are the representatives for IHC staining of tumor sections obtained from DU145 orthotopic-injected mouse using antibodies against EMT-related proteins (E-Cadherin, Vimentin) and cell stemness-related proteins (CD133, Nanog, Sox2, Oct4). Original magnification, × 100; scale bar, 100 μm. (M) The graph of the statistical results of (L), * P <0.05 v.s. stable-sh-Ctl, n = 6. (N-R) Images are the representatives (N, Q) and statistical results (O, P, R) for CD31/PAS double staining and IHC staining of tumor sections obtained from LNCaP orthotopic-injected mouse. Original magnification, × 100; scale bar, 100 μm. * P <0.05 v.s. stable-Ctl, n = 6.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: In Vivo, Imaging, Injection, Stable Transfection, Negative Control, shRNA, Plasmid Preparation, Over Expression, Double Staining, Immunohistochemistry

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: The screening of the downstream signal pathway involved in Wnt5a-induced VM formation was conducted. (A) Western blot analysis was used to quantify the expression levels of Vimentin, CD133, Nanog, Sox2, OCT4, VE-cadherin, and MMP9 in stable-Ctl LNCaP and stable-Wnt5a LNCaP cells treated as indicated. SP600125, JNK inhibitor; U-73,122, PLC inhibitor; TFA, PKG inhibitor; LF3, β-Catenin inhibitor; HSD1590, ROCK inhibitor. (B, C) Representative flow plots (B) and statistical chart (C) of CD133-positive population in LNCaP of different groups as indicated are shown. (D) Images of VM formation in 3D-cultured LNCaP is shown. Original magnification, × 200; scale bar, 200 μm. (E) The number of completely formed tubes was quantitated in (D). * P <0.05 v.s. as indicated, n = 3. (F) The expression of genes as indicated in DU145 was detected by qPCR. * P <0.05 v.s. as indicated, n = 3. (G-H) Representative (G) and statistical (H) images of VM formation in 3D-cultured DU145 is shown. Original magnification, × 200; scale bar, 200 μm. P <0.05 v.s. as indicated, n = 3. (I) The expression of genes as indicated in PC3 was detected by qPCR. * P <0.05 v.s. as indicated, n = 3. (J-K) Representative images (J) and statistical graph (K) of VM formation in 3D-cultured PC3 is shown. Original magnification, × 200; scale bar, 200 μm. P <0.05 v.s. as indicated, n = 3.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Western Blot, Expressing, Cell Culture

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: Wnt5a activates the JNK/c-jun pathway and promotes VM formation in a JNK-dependent manner. (A) The phosphorylation levels of JNK and c-jun were detected by western blot in stable-sh-Ctl DU145 and stable-shWnt5a DU145 cells. (B) The histogram showing the statistical results of gray values in (A), * P <0.05 v.s. stable-sh-Ctl, n = 3. (C, D) Immunoblotting analysis of phosphorylation level of JNK and c-jun in stable-Ctl LNCaP and stable-Wnt5a LNCaP cells. (D) is the statistical results of gray values of (C), * P <0.05 v.s. stable-Ctl, n = 3. (E) Representative images of immunohistochemical staining of tumor sections obtained from DU145 orthotopic-injected mice using antibodies against p-JNK and p-c-jun are shown. Original magnification, × 100; scale bar, 100 μm. (F) The statistical results of (E), * P <0.05 v.s. stable-sh-Ctl, n = 6. (G) The phosphorylation level of JNK and c-jun in tumor sections obtained from LNCaP orthotopic-injected mouse were evaluated by IHC staining. Original magnification, × 100; scale bar, 100 μm. (H) The statistical results of (G), * P <0.05 v.s. stable-Ctl, n = 6. (I) The expression of Vimentin, CD133, Nanog, Sox2, VE-cadherin and MMP9 in stable-sh-Ctl DU145 and stable-shWnt5a DU145 cells treated as indicated was quantified by western Blot. Vector, Empty vector plasmid; JNK-OV, JNK overexpression plasmid. (J-O) These histograms showing the statistical results of gray values of (I), * P <0.05 v.s. as indicated, n = 3. (P, Q) Representative flow cytometry diagrams (P) and statistical graph (Q) of CD133-positive in DU145 of different groups as indicated are shown, * P <0.05 v.s. as indicated, n = 3. (R, S) Representative images (R) and statistical graph (S) of tube formation in DU145 treated as indicated, Original magnification, × 200; scale bar, 200 μm; * P <0.05 v.s. as indicated, n = 3.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Western Blot, Immunohistochemical staining, Staining, Injection, Immunohistochemistry, Expressing, Plasmid Preparation, Over Expression, Flow Cytometry