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Stimulation of hepatocarcinogenesis by neutrophils upon induction of oncogenic kras expression in transgenic zebrafish.

Yan C, Huo X, Wang S, Feng Y, Gong Z - J. Hepatol. (2015)

Bottom Line: Both oncogenic hepatocytes and tumor-associated neutrophils (TANs) were isolated via fluorescence-activated cell sorting.Molecular analyses indicated a pro-inflammatory microenvironment, as marked by increased tgfβ1a expression in kras(V12)-expressing hepatocytes and a loss of anti-tumor activities in TANs.Depletion of Tgf-β significantly reduced the number of TANs and the size of oncogenic liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore; National University of Singapore Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore.

No MeSH data available.


Related in: MedlinePlus

Effect of antibody-mediated Tgf-β depletion on TAN recruitment. (A) Representative images of 8 dpf kras+/lyz+ larvae exposed to either doxycycline alone (top) or with SB431542 (bottom). (B) Neutrophil counts (left) and density (middle) in the liver and liver size (right) after SB431542 treatment. (C) Representative images of 8 dpf kras+/lyz+ larvae injected with Tgf-β antibody (bottom) or mock injected as a control (top). Both were induced by doxycycline. (D) Neutrophil counts (left) and density (middle) in the liver and liver size (right) in Tgf-β-depleted (tgfb-1D) and mock injected (MI) larvae. (E) Validation of SB431542 exposure and Tgf-β depletion by immunostaining of Smad2 and phospho-Smad2 (pSmad2). Representative liver sections are shown from each group as indicated. The color code of each probes correspond the color signals in the images. Red arrows, DsRed expressing neutrophils with pSmad2 expression; white arrows, DsRed expressing neutrophils without pSmad2 expression. (F) Ratios of pSmad2/smad2 in neutrophils (top) and hepatocytes (bottom) in SB431542-inhibited and Tgf-β depleted larvae. n = 10; *p <0.05.
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f0040: Effect of antibody-mediated Tgf-β depletion on TAN recruitment. (A) Representative images of 8 dpf kras+/lyz+ larvae exposed to either doxycycline alone (top) or with SB431542 (bottom). (B) Neutrophil counts (left) and density (middle) in the liver and liver size (right) after SB431542 treatment. (C) Representative images of 8 dpf kras+/lyz+ larvae injected with Tgf-β antibody (bottom) or mock injected as a control (top). Both were induced by doxycycline. (D) Neutrophil counts (left) and density (middle) in the liver and liver size (right) in Tgf-β-depleted (tgfb-1D) and mock injected (MI) larvae. (E) Validation of SB431542 exposure and Tgf-β depletion by immunostaining of Smad2 and phospho-Smad2 (pSmad2). Representative liver sections are shown from each group as indicated. The color code of each probes correspond the color signals in the images. Red arrows, DsRed expressing neutrophils with pSmad2 expression; white arrows, DsRed expressing neutrophils without pSmad2 expression. (F) Ratios of pSmad2/smad2 in neutrophils (top) and hepatocytes (bottom) in SB431542-inhibited and Tgf-β depleted larvae. n = 10; *p <0.05.

Mentions: Since Tgf-β has been reported to be a chemoattractant for neutrophils [38], we further depleted Tgf-β by injection of zebrafish Tgf-β antibody into 3 dpf kras+/lyz+ zebrafish as well as by SB431542 inhibition of TGF-β receptor [39]. We noticed reduced neutrophils in the liver by 8 dpf by both approaches (Fig. 8A and C) as confirmed by the decreases in neutrophil counts and density in the liver, which was also accompanied with a decrease in liver size (Fig. 8B and D). The successful depletion of Tgf-β was supported by significant reductions of phosphorylated Smad2, a downstream marker of the Tgf-β pathway in both neutrophils and hepatocytes (Fig. 8E–F). Collectively, these experiments demonstrate the role of Tgf-β in attracting neutrophils to the liver upon oncogenic kras activation in hepatocytes.


Stimulation of hepatocarcinogenesis by neutrophils upon induction of oncogenic kras expression in transgenic zebrafish.

Yan C, Huo X, Wang S, Feng Y, Gong Z - J. Hepatol. (2015)

Effect of antibody-mediated Tgf-β depletion on TAN recruitment. (A) Representative images of 8 dpf kras+/lyz+ larvae exposed to either doxycycline alone (top) or with SB431542 (bottom). (B) Neutrophil counts (left) and density (middle) in the liver and liver size (right) after SB431542 treatment. (C) Representative images of 8 dpf kras+/lyz+ larvae injected with Tgf-β antibody (bottom) or mock injected as a control (top). Both were induced by doxycycline. (D) Neutrophil counts (left) and density (middle) in the liver and liver size (right) in Tgf-β-depleted (tgfb-1D) and mock injected (MI) larvae. (E) Validation of SB431542 exposure and Tgf-β depletion by immunostaining of Smad2 and phospho-Smad2 (pSmad2). Representative liver sections are shown from each group as indicated. The color code of each probes correspond the color signals in the images. Red arrows, DsRed expressing neutrophils with pSmad2 expression; white arrows, DsRed expressing neutrophils without pSmad2 expression. (F) Ratios of pSmad2/smad2 in neutrophils (top) and hepatocytes (bottom) in SB431542-inhibited and Tgf-β depleted larvae. n = 10; *p <0.05.
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Related In: Results  -  Collection

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f0040: Effect of antibody-mediated Tgf-β depletion on TAN recruitment. (A) Representative images of 8 dpf kras+/lyz+ larvae exposed to either doxycycline alone (top) or with SB431542 (bottom). (B) Neutrophil counts (left) and density (middle) in the liver and liver size (right) after SB431542 treatment. (C) Representative images of 8 dpf kras+/lyz+ larvae injected with Tgf-β antibody (bottom) or mock injected as a control (top). Both were induced by doxycycline. (D) Neutrophil counts (left) and density (middle) in the liver and liver size (right) in Tgf-β-depleted (tgfb-1D) and mock injected (MI) larvae. (E) Validation of SB431542 exposure and Tgf-β depletion by immunostaining of Smad2 and phospho-Smad2 (pSmad2). Representative liver sections are shown from each group as indicated. The color code of each probes correspond the color signals in the images. Red arrows, DsRed expressing neutrophils with pSmad2 expression; white arrows, DsRed expressing neutrophils without pSmad2 expression. (F) Ratios of pSmad2/smad2 in neutrophils (top) and hepatocytes (bottom) in SB431542-inhibited and Tgf-β depleted larvae. n = 10; *p <0.05.
Mentions: Since Tgf-β has been reported to be a chemoattractant for neutrophils [38], we further depleted Tgf-β by injection of zebrafish Tgf-β antibody into 3 dpf kras+/lyz+ zebrafish as well as by SB431542 inhibition of TGF-β receptor [39]. We noticed reduced neutrophils in the liver by 8 dpf by both approaches (Fig. 8A and C) as confirmed by the decreases in neutrophil counts and density in the liver, which was also accompanied with a decrease in liver size (Fig. 8B and D). The successful depletion of Tgf-β was supported by significant reductions of phosphorylated Smad2, a downstream marker of the Tgf-β pathway in both neutrophils and hepatocytes (Fig. 8E–F). Collectively, these experiments demonstrate the role of Tgf-β in attracting neutrophils to the liver upon oncogenic kras activation in hepatocytes.

Bottom Line: Both oncogenic hepatocytes and tumor-associated neutrophils (TANs) were isolated via fluorescence-activated cell sorting.Molecular analyses indicated a pro-inflammatory microenvironment, as marked by increased tgfβ1a expression in kras(V12)-expressing hepatocytes and a loss of anti-tumor activities in TANs.Depletion of Tgf-β significantly reduced the number of TANs and the size of oncogenic liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore; National University of Singapore Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore.

No MeSH data available.


Related in: MedlinePlus