Journal: Journal of Extracellular Vesicles

Article Title: Perivascular cell‐derived extracellular vesicles stimulate colorectal cancer revascularization after withdrawal of antiangiogenic drugs

doi: 10.1002/jev2.12096

Figure Lengend Snippet: AA‐TKI withdrawal‐induced tumor revascularization is reduced after depletion of α‐SMA + or PDGFRβ + perivascular cells. (a) Therapeutic schedule for treatment with vehicle, regorafenib, and regorafenib + imatinib. Tumor vasculatures were analyzed at days 7 (Day 7) and 14 (withdrawal 7 d) after the indicated treatments. Withdrawal 7 d, 7 days after AA‐TKI withdrawal. (b) Representative images of CD31 + (red) endothelial cell, α‐SMA + (green) perivascular cell immunostaining, and DAPI (blue) for nuclear staining. Reg. regorafenib. (c) Quantification of microvessel density (CD31 density) and perivascular cell number (α‐SMA density) in HT‐29 tumors (n = 6). Veh., vehicle. W‐7 d, 7 days after AA‐TKI withdrawal. (d) Therapeutic schedule for treatment with vehicle + IgG, regorafenib + IgG, and regorafenib + anti‐PDGFRβ. Tumor vasculatures were analyzed at days 7 (Day 7) and 14 (withdrawal 7 d) after the indicated treatments. (e) Representative images of CD31 + (red) endothelial cell, PDGFRβ + (green) perivascular cell immunostaining, and DAPI (blue) for nuclear staining. (f) Quantification of microvessel density (CD31 density) and perivascular cell number (PDGFRβ density) in HT‐29 tumors (n = 6). Data are present as mean ± SEM. Scale bars, 50 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Antiangiogenic TKIs regorafenib and axitinib (TargetMol) suspended in 0.5% CMC‐Na solution (Selleck Chemicals) were used for in vivo studies.

Techniques: Immunostaining, Staining

Journal: Journal of Extracellular Vesicles

Article Title: Perivascular cell‐derived extracellular vesicles stimulate colorectal cancer revascularization after withdrawal of antiangiogenic drugs

doi: 10.1002/jev2.12096

Figure Lengend Snippet: Reduced tumor revascularization after ablation of NG2 + perivascular cells. (a) Therapeutic schedule for treatment with vehicle and regorafenib. Subcutaneous inoculation of Colon26 cancer cells in NG2‐tk mice and WT littermates, with daily GCV (50 mg/kg) or saline injections when tumors reached approximately 200 mm 3 . Tumor vasculatures were analyzed at days 7 (Day 7) and 12 (withdrawal 5 d) after the indicated treatments. Withdrawal 5 d, 5 days after treatment withdrawal. (b) Representative images of CD31 + (red) endothelial cell, NG2 + (green) perivascular cell immunostaining, and DAPI (blue) for nuclear staining. Scale bars, 50 μm. (c) Quantification of microvessel density (CD31 density) and perivascular cell number (NG2 density) in Colon26 tumors (n = 6). Data are present as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Antiangiogenic TKIs regorafenib and axitinib (TargetMol) suspended in 0.5% CMC‐Na solution (Selleck Chemicals) were used for in vivo studies.

Techniques: Saline, Immunostaining, Staining

Journal: Journal of Extracellular Vesicles

Article Title: Perivascular cell‐derived extracellular vesicles stimulate colorectal cancer revascularization after withdrawal of antiangiogenic drugs

doi: 10.1002/jev2.12096

Figure Lengend Snippet: Tumor perivascular cells regulate EPC recruitment into tumors during the revascularization phase. (a and b) Quantification of the proportional changes of recruited EPCs in HT‐29 tumors (n = 6). Withdrawal 3/7 d, 3/7 days after AA‐TKI withdrawal. W‐3/7 d, 3/7 days after AA‐TKI withdrawal. (c) Quantification of the proportional changes of recruited EPCs in Colon26 tumors from NG2‐tk+GCV and WT+GCV mice after the indicated treatments (n = 6). (d) Pearson correlation analysis of EPC proportion and CD31 density in (d, left) HT‐29 tumors after treatment with AA‐TKIs for 7 days or withdrawal for 7 days; or in (d, right) Colon26 tumors from WT+GCV mice after treatment with regorafenib for 7 days or withdrawal for 5 days. (e) Pearson correlation analysis of (e, left) EPC proportion and perivascular cell number (α‐SMA density) in HT‐29 tumors after treatment with AA‐TKIs for 7 days or withdrawal for 7 days, or (e, right) EPC proportion and perivascular cell number (NG2 density) in Colon26 tumors from WT+GCV and NG2‐tk+GCV mice after treatment with regorafenib for 7 days or withdrawal for 5 days. (f) Representative images of PKH67‐labeled EPCs (green) recruited into HT‐29 tumors (mice 1) and Colon26 tumors (mice 2). DAPI (blue) was used for nuclear staining. (g) HT‐29 tumor‐bearing mice were treated with regorafenib + anti‐PDGFRβ for 7 days or (H) Colon26 tumor‐bearing NG2‐tk+GCV mice were treated with regorafenib for 7 days, followed by tail vein injection of 5 × 10 5 EPCs every other day. Tumor microvessel density was determined after 5 days. Representative images of CD31 + (red) endothelial cell, PDGFRβ + or NG2 + (green) perivascular cell immunostaining, and DAPI (blue) for nuclear staining. Quantification of CD31 density (n = 6). Data are present as mean ± SEM. Scale bars, 50 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Antiangiogenic TKIs regorafenib and axitinib (TargetMol) suspended in 0.5% CMC‐Na solution (Selleck Chemicals) were used for in vivo studies.

Techniques: Labeling, Staining, Injection, Immunostaining

Journal: Journal of Extracellular Vesicles

Article Title: Perivascular cell‐derived extracellular vesicles stimulate colorectal cancer revascularization after withdrawal of antiangiogenic drugs

doi: 10.1002/jev2.12096

Figure Lengend Snippet: TPC‐EVs promote EPC recruitment into tumors to facilitate tumor revascularization. (a‐c) The characterization and quantification of TPC‐EVs by (a) TEM, (b) particle size, and (c) Western blotting analyses. (d) USPIO‐loaded TPC‐EVs (100 μg) were intravenously injected into mice and TPC‐EV incorporation into bone marrow was detected by magnetic resonance imaging analysis. White arrows indicated the accumulation of USPIO‐loaded TPC‐EVs. USPIO, ultrasmall superparamagnetic iron oxide nanoparticles. (e) PKH26‐labeled TPC‐EVs (100 μg) were intravenously injected into mice. Barrow cells were harvested after 24 h and then fixed and incubated with red blood cell lysis buffer. Representative images of incorporated TPC‐EVs (red) in marrow cells. Scale bar, 20 μm. (f) HT‐29 tumor‐bearing mice received the treatment of regorafenib or axitinib for 7 days, followed by tail vein injection with PBS (Vehicle) or TPC‐EVs (100 μg) for 4 days. Then, EPC recruitment and tumor vasculature were analyzed. (g) Quantification of the proportional changes of recruited EPCs in HT‐29 tumors (n = 6). (h) Representative images of CD31 + (red) endothelial cell, α‐SMA + (green) perivascular cell immunostaining, and DAPI (blue) for nuclear staining. Scale bar, 50 μm. (i) Quantification of microvessel density (CD31 density) and perivascular cell number (α‐SMA density) in HT‐29 tumors (n = 6). Colon26 tumor‐bearing NG2‐tk+GCV mice were treated with regorafenib for 7 days, followed by tail vein injection of Vehicle or TPC‐EVs (100 μg) for 4 days. (j) Quantification of the proportional changes of recruited EPCs in Colon26 tumors (n = 6). (k) Representative images of CD31 + (red) endothelial cell, NG2 + (green) perivascular cell immunostaining, and DAPI (blue) for nuclear staining. Scale bar, 50 μm. (l) Quantification of microvessel density (CD31 density) and perivascular cell number (NG2 density) in Colon26 tumors (n = 6). Data are present as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Antiangiogenic TKIs regorafenib and axitinib (TargetMol) suspended in 0.5% CMC‐Na solution (Selleck Chemicals) were used for in vivo studies.

Techniques: Western Blot, Injection, Magnetic Resonance Imaging, Labeling, Incubation, Red Blood Cell Lysis, Immunostaining, Staining

Journal: Journal of Extracellular Vesicles

Article Title: Perivascular cell‐derived extracellular vesicles stimulate colorectal cancer revascularization after withdrawal of antiangiogenic drugs

doi: 10.1002/jev2.12096

Figure Lengend Snippet: Inhibition of the Axl pathway impairs EPC recruitment and suppresses tumor revascularization after AA‐TKI treatment. (a) Therapeutic schedule for R428 administration in HT‐29 tumor‐bearing mice after regorafenib or axitinib treatment. (b) Quantification of the proportional changes of recruited EPCs in HT‐29 tumors (n = 6). (c) Representative images of CD31 + (red) endothelial cell, α‐SMA + (green) perivascular cell immunostaining, and DAPI (blue) for nuclear staining. (d) Quantification of microvessel density (CD31 density) and perivascular cell number (α‐SMA density) in HT‐29 tumors (n = 6). (e) Representative images of cells with p‐Histone H3 positive staining (green) and DAPI (blue) for nuclear staining. Quantification of cell proliferation (p‐Histone H3 index) in HT‐29 tumors (n = 6). Data are presented as mean ± SEM. Scale bars, 50 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Antiangiogenic TKIs regorafenib and axitinib (TargetMol) suspended in 0.5% CMC‐Na solution (Selleck Chemicals) were used for in vivo studies.

Techniques: Inhibition, Immunostaining, Staining

Journal: Journal of Extracellular Vesicles

Article Title: Perivascular cell‐derived extracellular vesicles stimulate colorectal cancer revascularization after withdrawal of antiangiogenic drugs

doi: 10.1002/jev2.12096

Figure Lengend Snippet: Regorafenib plus R428 therapy prolongs survival and decreases the tumor burden in mice with colorectal cancer liver metastases. (a) Therapeutic schedule for continuous treatment with vehicle, regorafenib, R428, and regorafenib + R428 in mice bearing HCT116 liver metastases. (b) Kaplan‐Meier estimates of overall survival for mice with HCT116 hepatic tumors after the indicated treatment (n = 7). OS, overall survival. MS, median survival. Reg., regorafenib. NS, no significance. * P < 0.05 and ** P < 0.01 versus indicated groups by Log‐rank (Mantel‐Cox) test. (c) Representative ultrasound images of HCT116 hepatic tumors after the indicated treatments. Mice bearing HCT116 hepatic tumors were treated as indicated and imaged at days 15 and 28 after tumor implantation. Scale bar, 5 mm. (d) Representative images of CD31 + (red) endothelial cell immunostaining and DAPI (blue) for nuclear staining. Scale bar, 50 μm. Quantification of microvessel density (CD31 density) in HCT116 hepatic tumors (n = 8). Data are presented as mean ± SEM. *** P < 0.001. (e) Diagram depicting the mechanism by which Gas6 + TPC‐EVs activates the Axl pathway and consequently promotes EPC recruitment for rapid tumor revascularization after withdrawal of AA‐TKI therapy

Article Snippet: Antiangiogenic TKIs regorafenib and axitinib (TargetMol) suspended in 0.5% CMC‐Na solution (Selleck Chemicals) were used for in vivo studies.

Techniques: Tumor Implantation, Immunostaining, Staining