Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 1 | The expression of ENO1 on the cell-surface of a subpopulation of CSCs. (A) Representative FACS plots showing patterns of CD44, CD133, and surface ENO1 (sENO1) staining of primary prostate adenocarcinoma (PAC)-derived 22Rv-1 cells with the frequency of the boxed CD44+CD133+ cell population (representing CSCs in PAC; left) or sENO1+ cells in CD44+CD133+ CSCs (middle) or cells in the other subpopulations (representing non-CSCs; right) shown. (B) The percentages of sENO1+ cell subpopulation in CD44+CD133+ 22Rv-1 cells or cells in the other subpopulations (others). (C) The percentages of sENO1+ cell subpopulation in CD44+CD133+ PC-3 cells or cells in the other subpopulations. (D) The percentages of sENO1+ cell subpopulation in CD90+ gastric adenocarcinoma (GAC) AGS or NCI-N87 cells (representing CSCs in GAC) or CD90- cells (representing non-CSCs). Error bars represent mean ± SEM from three independent experiment (n = 3). Unpaired t-test was performed throughout where **p < 0.01; ***p < 0.001 in (B–D).

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Expressing, Staining, Derivative Assay

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 2 | sENO1 marks a subpopulation of mesenchymal-like and highly invasive CSCs. (A) The relative transcript levels of the mesenchymal- (left) or pluripotency- (right) associated genes in sENO1+ CSCs (represented by CD44+CD133+ 22Rv-1 cells), sENO1- CSCs, and non-CSCs (represented by cells in the other subpopulations) using qRT-PCR analysis. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where *p < 0.05 versus non-CSCs; †p < 0.05 versus sENO1- CSCs. (B) Immunoblotting analysis of the indicated markers selected from (A) in non- CSCs, sENO+, and sENO- CSCs. Protein levels were quantified by densitometric analysis of the bands, normalized to b-tubulin (loading control). (C) Limiting dilution assay (LDA) demonstrating the tumorsphere-forming efficacy of each subset of tumor cells. Three independent experiments were performed (n = 6). Shown are maximum likelihood estimates with a 95% confidence interval, where **p < 0.01. (D) The invasive capacities of freshly sorted sENO1+ CSCs (represented by CD44+CD133+ 22Rv-1 cells), sENO1- CSCs, and non-CSCs in 22Rv-1 cells in a dual-chamber invasion assay. Shown are representative immunofluorescence images of the invaded cells, with cell nuclei stained with SYTOX-green (green). Scale bars = 500 µm. Right, the number of invaded cells. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where **p < 0.01.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Quantitative RT-PCR, Western Blot, Control, Limiting Dilution Assay, Invasion Assay, Staining

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 3 | sENO1+ CSCs are highly pro-metastatic. (A) Representative BLI of NOD/SCID mice receiving an intra-splenic injection of sENO1+ CSCs (represented by CD90+ NCI-N87 cells), sENO1- CSCs (CD90- NCI-N87 cells), and non-CSCs (represented by CD90- cells). at the indicated time following cell inoculation. (B) Tumor bulk quantified as BLI normalized photon counts as a function of time. Error bars represent mean ± SEM from one experiment (n = 8 mice per group). Unpaired t-test was performed throughout where **p < 0.01 versus non-CSCs. (C) Representative BLI of NOD/SCID mice receiving intra-splenic injection of sENO1+

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Injection

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 4 | ENO1 is expressed on the invadopodial surface of CSCs. (A) Confocal views of PAC CSCs (represented by CD44+CD133+ PC-3 cells) showing the cross-section of invadopodia structures (represented by cortactin+F-acin+ puncta) with the colocalized surface ENO1 (sENO1; green), cortactin (red), and F-actin (magenta) that penetrate into the underlying gelatin matrix. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale, 10 µm. (B) Top, a pie chart showing the percentage of sENO1+ invadopodia per PC-3 CSC. Bottom, a pie chart showing the percentage of sENO1+ invadopodia per GAC AGS CSC (represented by CD90+ AGS cells). (C) Left, representative three-dimensional (3D) reconstructed confocal image of CD44+CD133+ PC-3 CSCs showing the co- localization of sENO1 (green) and cortactin (red) at the ventral side of cell. Scale, 8 µm. Right upper, digital zoom-in image from serial Z sections (yellow rectangle) showing the spatial colocalization of sENO1 (green) and cortactin (red) at invadopodia. Scale, 5 µm. Right lower, the orthogonal view of the magnified areas (yellow squares at top) shown the distribution and localization of sENO1 and cortactin at the base of invadopodia. 3D rendered images of the invadopodia (arrows) were processed by using Imaris software. Scale, 1 µm.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Software

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 5 | sENO1+ CSCs generate more invadopodia than their sENO1- counterparts. (A) Confocal views of sENO1+ PC-3 CSCs (represented by CD44+CD133+

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques:

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 6 | sENO1 contributes to the invadopodial formation and the matrix-degradative function of CSCs. (A) Immunoblotting analysis showing the effect of lentivirus shRNA-mediated knockdown (KD) of ENO1 expression in PC-3 cells. Protein levels were quantified by densitometric analysis of the bands, normalized to b- tubulin (loading control). (B) Bar graph showing the percentage of sENO1+ PC-3 cells with KD of ENO1 expression or control KD. (C) Bar graph showing the density of invadopodia (represented by cortactin+F-actin+ puncta) per cells in PC-3 CSCs (represented by CD44+CD133+ cells) or non-CSCs (represented by cells in other subpopulations) with ENO1 KD or control KD. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed where **p < 0.01, ***p < 0.001 in (B, C). (D) PC-3 cells with KD of ENO1 expression or the control KD cells were seeded on top of a fluorescein-conjugated gelatin matrix and immunostained with cortactin (green) or phalloidin (F-actin; red). Nuclei were counterstained with DAPI (blue). Right, the fluorescence intensity of fluorescein- conjugated gelatin within the boundary (determined by F-actin staining) of PC-3 cells with ENO1 KD or control KD (n = 50 cells counted per sample). Unpaired t-test was performed where ***p < 0.001. (E) Bar graph showing the invasive capacity of PC-3 CSCs with ENO1 KD or control KD in a dual-chamber invasion assay. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed where **p < 0.01 versus non-CSCs. (F) Representative immunofluorescence images of CD44+CD133+ PC-3 cells (representing CSCs) that had invaded the type I collagen matrix in the presence of an increasing concentration (0.1-1.0 µg/ml) of the anti-ENO1 polyclonal antibody (pAb; a-ENO1) in a dual-chamber invasion assay. The nuclei of the invaded cells were stained with SYTOX-green. Scale bars, 500 µm. Right, the number of invaded cells. Cells in other subpopulations (representing non-CSCs) were included as a control. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where *p < 0.05, **p < 0.01 versus non-CSCs. (G) The invadopodia density per cell in PC-3 CSCs or non-CSCs exposed to an increasing concentration of a-ENO1. Error bars represent mean ± SEM from three independent experiments (n = 50 cells counted per sample). Unpaired t-test was performed where *p < 0.05, **p < 0.01, ***p < 0.001 versus non-CSCs. (H) PC-3 CSCs were seeded on top of a gelatin matrix in the presence or absence of a-ENO1 (20 µg/ml). Shown are the extent of matrix degradation as reflected by immunostaining with anti-Col1-3/4C (red). Right, the total cell fluorescence intensity of Col1-3/4C in PC-3 CSCs treated with a-ENO1 or a control IgG (n = 50 cells counted per sample). Unpaired t-test was performed where ***p < 0.001.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Western Blot, shRNA, Knockdown, Expressing, Control, Staining, Invasion Assay, Concentration Assay, Immunostaining

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 7 | CAV1 is indispensable for the surface localization of sENO1 on CSCs and its pro-invadopodia and pro-invasive functions. (A) Immunoblotting analysis showing the effect of lentivirus shRNA-mediated knockdown (KD) of CAV1 (top) or HSP70 (bottom) expression in PC-3 cells. Protein levels were quantified by densitometric analysis of the bands, normalized to b-tubulin (loading control). (B) Bar graph showing the percentage of sENO1+ cells in PC-3 CSCs (represented by CD44+CD33+ cells) with KD of CAV1 or HSP70 expression or control-KD. Unpaired t-test was performed throughout where ***p < 0.001 versus control KD. (C) Bar graph showing the density of invadopodia (represented by coractin+F-actin+ puncta) per cell in PC-3 CSCs or non-CSCs (represented by cells in other subpopulations) with CAV1 KD or control KD. Unpaired t-test was performed throughout where ***p < 0.001. (D) Bar graph showing the invasive capacity of PC-3 CSCs or non-CSCs with CAV1 KD or control KD in a dual-chamber invasion assay. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where *p < 0.05 versus non-CSCs; †p < 0.05 versus control KD.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Western Blot, shRNA, Knockdown, Expressing, Control, Invasion Assay