Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 1. Upregulation of ALDH1A3 in oxGBM cells is associated with the increased expression and release of pro-angiogenic factors PAI-1 and IL-8. GBM cell lines were transduced with ALDH1A3 for overexpression (oxGBM) or with empty vector (evGBM). (A) Confirmation of up-regulation of ALDH1A3 mRNA level in oxGBM cells by RT2-PCR. (B) Confirmation of up-regulation of ALDH1A3 protein expression by Western blot. wt, wild-type cells. The uncropped blots are shown in Figure S7; (C) Angiogenesis array. The blots showed duplicated dots for 55 angiogenesis-related proteins in the media of oxU373 or evU373. 10 of 55 proteins were upregulated more than 2-fold in ox group compared to ev group (indicated by rectangle). They are: (1) Ang-1, (2) artemin, (3) TF, (4) ET-1, (5) GM-CSF, (6) IL-8, (7) PDGF-AA, (8) PAI-1, (9) PEDF, and (10) uPA. (D) Semi-quantification of the dots representing PAI-1 and IL-8. (E) Immunofluorescence staining of GBM cells. U373 (left panel) and LN229 (right panel). Co-localization of ALDH1A3 with PAI-1 and IL-8 was observed in oxGBM cells, whereas no immunoreactivity of PAI-1 and IL-8 was detected in evGBM cells. (F) mRNA expression of PAI-1 and IL-8 in transduced U373 cells and the effect of inhibitors. Tiplaxtinin (Tip, 30 µM) and reparixin (Rep, 1 µM) are the specific inhibitors of PAI-1 and IL-8 receptors CXCR1/2, respectively. (G) Detection of PAI-1 and potential signaling proteins by Western blot. IOD: optical density. The uncropped blots are shown in Figure S8. **, p < 0.01; ***, p < 0.001, compared with ev. ##, p < 0.01; ###, p < 0.001, compared with ox.

Article Snippet: To determine the effects of GBM-derived PAI-1 and IL-8 on EC behavior, the following inhibitors were used: Tiplaxtinin (cat# HY-15253; MedChemExpress, Monmouth Junction, NJ, USA), a small-molecule inhibitor of PAI-1, and reparixin (cat# HY-15251; Med-ChemExpress), an allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2.

Techniques: Expressing, Transduction, Over Expression, Plasmid Preparation, Western Blot, Staining

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 2. Overexpression of ALDH1A3 in GBM cells activated endothelial angiogenesis in indirect co-culture with endothelial cells (ECs), which was reversed by treatment with respective inhibitors of PAI-1 or IL-8 receptors CXCR1/2. Indirect co-culture was performed by culture of HBMECs in a conditioned medium (CM) containing the media derived from evGBMs or oxGBMs and ECGM in a ratio of 1:1. PAI-1 inhibitor tiplaxtinin (Tip, 30 µM) and CXCR1/2 inhibitor reparixin (Rep, 1 µM) or vehicle DMSO (0.1%) was added to CM, followed by EC behavior study. All data were reproduced in three independent experiments. (A) Proliferation assay in HBMEC and HUVEC. Indirect co-culture of HBMECs and HUVECs with CM derived from oxU373 and oxLN229 stimulated EC proliferation, which was completely reversed by the treatment of tiplaxtinin, not by reparixin. (B) Scratch assay in HBMEC. Left panel: images were acquired 24 h after scratching. Scale bar: 200 µm. Right panel: quantitative analysis. Culture of HBMECs with CM derived from oxU373 or oxLN229 (oxCM) significantly promoted HBMEC migration, which was reversed by the treatment of tiplaxtinin and reparixin, respectively. (C) Transwell invasion assay in HBMEC. Left panel: Representative images of invaded cells were acquired after 24 h of incubation. Scale bar: 100 µm. Right panel: quantitative analysis. Culture of HBMECs with oxCM accelerated HBMEC invasion. This effect was significantly inhibited by the treatment of reparixin but not by tiplaxtinin. (D) Tube formation assay in HBMEC. Left panel: representative images of tube formation. Scale bar: 200 µm. Right panel: quantitative analysis of branching points per field. Tube formation in HBMECs was stimulated by incubation with oxCM, which was completely diminished by both inhibitors. (E) Sprouting assay in HBMEC. Left panel: representative images of sprouting in HBMECs after 24 h of co-culture. Scale bar: 100 µm. A pronounced increase in sprouting was observed in HBMECs cultured in oxCM. Tiplaxtinin and reparixin suppressed the sprouting effect resulting from oxCM. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, compared with evCM. #, p < 0.05; ##, p < 0.01 and ###, p < 0.001, compared with oxCM.

Article Snippet: To determine the effects of GBM-derived PAI-1 and IL-8 on EC behavior, the following inhibitors were used: Tiplaxtinin (cat# HY-15253; MedChemExpress, Monmouth Junction, NJ, USA), a small-molecule inhibitor of PAI-1, and reparixin (cat# HY-15251; Med-ChemExpress), an allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2.

Techniques: Over Expression, Co-Culture Assay, Derivative Assay, Proliferation Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Incubation, Tube Formation Assay, Cell Culture

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 3. Overexpression of ALDH1A3 in GBM cells stimulated EC proliferation, migration, and sprouting in direct co-culture with endothelial cells (ECs), which was inhibited by treatment with the specific inhibitors of PAI-1 or IL-8 receptors CXCR1/2. Direct co-culture was performed by co- culture of CellTrace™CFSE pre-labeled HBMECs with transduced U373 and LN229 in a ratio of 1:1 in ECGM. To inhibit PAI-1, tiplaxtinin (Tip, 30 µM) was pretreated with GBM cells 6 h in advance. For IL-8 receptor inhibition, reparixin (Rep, 1 µM) was pretreated with ECs 30 min in advance and added to ECGM in the co-culture, as well. All data were reproduced in three independent experiments. (A) Representative images of directly co-cultured CellTrace™CFSE-labeled HBMEC (green) with transduced U373 and LN229. Scale bar: 100 µm. (B) HBMEC proliferation. Direct co-culture of oxGBMs with HBMECs promoted the proliferation of HBMEC, which was completely reversed by the treatment of tiplaxtinin and reparixin. The fluorescence intensity of CFSE-labeled HBMECs, reflecting cell proliferation, was detected 48 h after direct co-culture at 485 nm of excitation wavelength and 535 nm of emission wavelength. (C) HBMEC migration. The images were acquired after 12 h of direct

Article Snippet: To determine the effects of GBM-derived PAI-1 and IL-8 on EC behavior, the following inhibitors were used: Tiplaxtinin (cat# HY-15253; MedChemExpress, Monmouth Junction, NJ, USA), a small-molecule inhibitor of PAI-1, and reparixin (cat# HY-15251; Med-ChemExpress), an allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2.

Techniques: Over Expression, Migration, Co-Culture Assay, Labeling, Inhibition, Cell Culture

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 5. Immunohistochemistry and immunofluorescence staining on GBM sections. (A,B) Im- munohistochemistry staining of ALDH1A3. The immunoreactivity of ALDH1A3 was detected in vessels (arrows in (A)) and peripheral cells of glomeruloid (arrowheads in (B)) as well as in tumor cells (asterisks in (A)). Scale bar: 50 µm in (A) and 20 µm in (B). (C–E) Double-immunofluorescence staining of ALDH1A3 (red) with PAI-1 (green) (C,D) or with IL-8 (green) (E,F). The co-localization of ALDH1A3 with PAI-1 or with IL-8 immunoreactivity was detected in microvessels (arrows), glomeruloid (arrowheads), and tumor cells (asterisks) in GBM sections.

Article Snippet: To determine the effects of GBM-derived PAI-1 and IL-8 on EC behavior, the following inhibitors were used: Tiplaxtinin (cat# HY-15253; MedChemExpress, Monmouth Junction, NJ, USA), a small-molecule inhibitor of PAI-1, and reparixin (cat# HY-15251; Med-ChemExpress), an allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2.

Techniques: Immunohistochemistry, Staining