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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

Recruitment of neutrophils to oncogenic liver. (A) Representative images of kras−/lyz+ and kras+/lyz+ larvae after 8, 24, and 96 hours of doxycycline induction starting from 3 dpf. The livers are outlined in blue dash lines in the upper images of kras−/lyz+ larvae and marked by GFP expression in the lower images of kras+/lyz+ larvae. (B–D) Time course of neutrophil count (B), neutrophil density (C) and liver size (D) following induction of oncogenic krasV12 expression in hepatocytes (n >15 from each group). Neutrophils were counted in the liver area and normalized against the liver size for liver density. Liver size was measured based on 2D images. (E) Correlation of liver size and neutrophil density in the liver. The measurements were based on 8 dpf larvae after 5 days of doxycycline induction. A positive correlation was observed only in the kras+ transgenic larvae (top) but not in the kras− control group (bottom). Statistical significance: *p <0.05, **p <0.01, ***p <0.001.
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f0005: Recruitment of neutrophils to oncogenic liver. (A) Representative images of kras−/lyz+ and kras+/lyz+ larvae after 8, 24, and 96 hours of doxycycline induction starting from 3 dpf. The livers are outlined in blue dash lines in the upper images of kras−/lyz+ larvae and marked by GFP expression in the lower images of kras+/lyz+ larvae. (B–D) Time course of neutrophil count (B), neutrophil density (C) and liver size (D) following induction of oncogenic krasV12 expression in hepatocytes (n >15 from each group). Neutrophils were counted in the liver area and normalized against the liver size for liver density. Liver size was measured based on 2D images. (E) Correlation of liver size and neutrophil density in the liver. The measurements were based on 8 dpf larvae after 5 days of doxycycline induction. A positive correlation was observed only in the kras+ transgenic larvae (top) but not in the kras− control group (bottom). Statistical significance: *p <0.05, **p <0.01, ***p <0.001.

Mentions: To visualize the inflammation response, hemizygous kras+ transgenic fish were crossed with lyz+ homozygous fish for production of kras+ and kras− offspring in the lyz+ background, DsRed expressing neutrophils were monitored up to 96 hpi (hour post-induction with doxycycline). As shown in Fig. 1A and quantified in Fig. 1B–D, the total counts of neutrophils in the liver region was noticeably increased from as early as 8 hpi and became statistically significant from 16 hpi (Fig. 1B). These neutrophils within the vicinity of the liver, considered as TANs, were normalized against the liver size as neutrophil density. As shown in Fig. 1C, a significant increase in neutrophil density was observed from 8 hpi; thus, neutrophils were actively recruited to the site of tumor initiation within 8 hours of oncogene activation. In contrast, the increase of liver size became apparent only from 24 hpi (Fig. 1D), indicating that neutrophil recruitment preceded liver enlargement. To further evaluate the contribution of neutrophils to the increased liver size in kras+ larvae, neutrophil density and liver size were plotted for each individual kras+ larva and we observed a strong positive correlation (Pearson’s coefficient, 0.62); in contrast, such a correlation was not present in kras− siblings (Pearson’s coefficient, 0.20) (Fig. 1E).


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)

Recruitment of neutrophils to oncogenic liver. (A) Representative images of kras−/lyz+ and kras+/lyz+ larvae after 8, 24, and 96 hours of doxycycline induction starting from 3 dpf. The livers are outlined in blue dash lines in the upper images of kras−/lyz+ larvae and marked by GFP expression in the lower images of kras+/lyz+ larvae. (B–D) Time course of neutrophil count (B), neutrophil density (C) and liver size (D) following induction of oncogenic krasV12 expression in hepatocytes (n >15 from each group). Neutrophils were counted in the liver area and normalized against the liver size for liver density. Liver size was measured based on 2D images. (E) Correlation of liver size and neutrophil density in the liver. The measurements were based on 8 dpf larvae after 5 days of doxycycline induction. A positive correlation was observed only in the kras+ transgenic larvae (top) but not in the kras− control group (bottom). Statistical significance: *p <0.05, **p <0.01, ***p <0.001.
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Related In: Results  -  Collection

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f0005: Recruitment of neutrophils to oncogenic liver. (A) Representative images of kras−/lyz+ and kras+/lyz+ larvae after 8, 24, and 96 hours of doxycycline induction starting from 3 dpf. The livers are outlined in blue dash lines in the upper images of kras−/lyz+ larvae and marked by GFP expression in the lower images of kras+/lyz+ larvae. (B–D) Time course of neutrophil count (B), neutrophil density (C) and liver size (D) following induction of oncogenic krasV12 expression in hepatocytes (n >15 from each group). Neutrophils were counted in the liver area and normalized against the liver size for liver density. Liver size was measured based on 2D images. (E) Correlation of liver size and neutrophil density in the liver. The measurements were based on 8 dpf larvae after 5 days of doxycycline induction. A positive correlation was observed only in the kras+ transgenic larvae (top) but not in the kras− control group (bottom). Statistical significance: *p <0.05, **p <0.01, ***p <0.001.
Mentions: To visualize the inflammation response, hemizygous kras+ transgenic fish were crossed with lyz+ homozygous fish for production of kras+ and kras− offspring in the lyz+ background, DsRed expressing neutrophils were monitored up to 96 hpi (hour post-induction with doxycycline). As shown in Fig. 1A and quantified in Fig. 1B–D, the total counts of neutrophils in the liver region was noticeably increased from as early as 8 hpi and became statistically significant from 16 hpi (Fig. 1B). These neutrophils within the vicinity of the liver, considered as TANs, were normalized against the liver size as neutrophil density. As shown in Fig. 1C, a significant increase in neutrophil density was observed from 8 hpi; thus, neutrophils were actively recruited to the site of tumor initiation within 8 hours of oncogene activation. In contrast, the increase of liver size became apparent only from 24 hpi (Fig. 1D), indicating that neutrophil recruitment preceded liver enlargement. To further evaluate the contribution of neutrophils to the increased liver size in kras+ larvae, neutrophil density and liver size were plotted for each individual kras+ larva and we observed a strong positive correlation (Pearson’s coefficient, 0.62); in contrast, such a correlation was not present in kras− siblings (Pearson’s coefficient, 0.20) (Fig. 1E).

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