Enhancement of mammary tumour growth by IGFBP-3 involves impaired T cell accumulation

1. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 1

   EO771 cells do not express detectable IGFBP-3 as determined by (A) mRNA expression compared to murine 4T1 breast cancer cells (normalised to 1.0) and mouse liver and (B) secreted IGFBP-3 in culture medium, measured by ELISA in comparison to mouse cardiac endothelial cells. Comparisons made against 4T1 and endothelial cells were conducted in triplicate. Data are presented as means ± s.e.m. (C) Increased intratumoural mRNA expression of Igfbp3 relative to Hmbs associated with increased tumour weight (n = 16 per group). Data were analysed by Spearman’s correlation test.
2. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 2

   Intratumoural T cell abundance is increased in the absence of IGFBP-3. (A) Gene expression of the T cell markers, Cd8a and Cd8b1, is increased in tumours from BP3KO mice but reduced by HFD (Cd8a: P < 0.0001 for genotype and P = 0.023 for diet, Cd8b1: P < 0.0002 for genotype and P = 0.008 for diet, n = 15–16 per group). Gene expression of the T cell marker, Cd4, is increased in tumours from high fat-fed as well as BP3KO mice (P = 0.035 for genotype and P = 0.02 for diet, n = 14–16 per group). (B and C) Cd8a and Cd8b1 gene expressions in the tumour are negatively associated with tumour weight across all groups. (D) The ratio of cells expressing Cd8b1:Cd4 mRNA is increased in chow-fed BP3KO mice (P = 0.028 for genotype and P < 0.001 for diet). (E) Combined gene expression of Cd8b1 and Cd4 in tumours from wild-type and BP3KO mice. Data were analysed by 2-way ANOVA or by Spearman’s correlation test and are shown as means ± s.e.m. Brackets indicate groups that are significantly different by post hoc Tukey’s test.
3. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 3

   (A) The proportion of CD8⁺ T cells, identified by immunohistochemistry, is increased in tumours from BP3KO mice and reduced by HFD (P = 0.008 for genotype and P = 0.032 for diet, n = 11–15 per group). (B) Cd8a gene expression is strongly associated with CD8 staining by IHC. (C, D, E and F) Representative images of tumour sections stained for CD8. Data were analysed by 2-way ANOVA or by Spearman’s correlation test and are shown as means ± s.e.m. Brackets indicate groups that are significantly different by post hoc Tukey’s test.
4. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 4

   Expression of markers associated with T cells and natural killer cells. (A) Intratumoural expression of Gzmb is not different overall between wild-type and BP3KO mice but is reduced with HFD in BP3KO mice (P = 0.024 for interaction). Intratumoural gene expression of the regulatory T cell marker, Foxp3, and the natural killer cell marker, Nkg2d, were not different among treatment groups. (B) Granzyme B expression was similar in tumours across groups by IHC detection. (C) Representative images of a tumour section stained positively for granzyme B and the associated isotype control-stained tumour section (inset). Gzmb expression in the tumours is (D) positively associated with Cd8b1 expression and (E) negatively associated with tumour weight across all groups. (F) Nkg2d expression is negatively associated with tumour growth. Data were analysed by 2-way ANOVA or by Spearman’s correlation test and are shown as means ± s.e.m., n = 15–16 per group. Brackets indicate groups that are significantly different by post hoc Tukey’s test.
5. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 5

   Cytokines associated with anti-tumour activity are increased in tumours from BP3KO mice. (A) Tumoural gene expressions of (A) Ifng and Tnf are increased in the absence of IGFBP-3 (Ifng: P = 0.008 and Tnf: P = 0.049 for genotype). Tumoural gene expression of Tnfsf10, which encodes TRAIL, is similar across treatment groups. (B) The increased expression of Ifng in BP3KO but not wild-type mice is associated with decreased tumour weight. (C) Increased expression of Tnf is associated with decreased tumour weight. (D) Expression of Tnfsf10 is positively associated with gene expression of the natural killer cell marker, Nkg2d, and (E) negatively associated with tumour weight. Data were analysed by 2-way ANOVA or by Spearman’s correlation test and are shown as means ± s.e.m., n = 15–16 per group. Brackets indicate groups that are significantly different by post hoc Tukey’s test.

• © 2018 Society for Endocrinology