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MDA-MB-231 breast cancer cells overexpressing single VEGF isoforms display distinct colonisation characteristics

View Article: PubMed Central - PubMed

ABSTRACT

Background:: Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that has important roles in angiogenesis. Our knowledge of the significance of VEGF isoforms in human cancer remains incomplete.

Methods:: Bioluminescence imaging and transcriptomic analysis were used to study the colonisation capacity of the human breast cancer cells MDA-MB-231 controlling or overexpressing the VEGF165 or VEGF189 isoform (named cV-B, V165-B and V189-B, respectively) in nude mice.

Results:: When injected into the bloodstream, V189-B cells induced less metastasis in the lungs and bone than V165-B and cV-B control cells, consistent with longer survival of these mice and delay in tumour uptake in the mice injected with a V189-B clone. Histological analysis confirmed that there were less αSMA-positive cells in the lungs of the mice injected with V189-B. In vitro V189-B cells decreased both cell invasion and survival. Using transcriptomic analysis, we identified a subset of 18 genes expressed differentially between V189 and V165 cell lines and in 120 human breast tumours. V165 was associated with poor prognosis, whereas V189 was not, suggesting a complex regulation by VEGF isoforms. Our results showed a negative correlation between the expression pattern of VEGF189 and the levels of expression of seven genes that influence metastasis.

Conclusion:: Our findings provide the first evidence that VEGF isoforms have different effects on breast cancer cell line colonisation in vivo.

No MeSH data available.


Related in: MedlinePlus

Stromal compartment and migration of gene expression. (A and B) Immunostaining (A) and quantification (B) for SM-alpha actin. For each animal, two sections per lung positive for tumour cells were immunostained with SM-alpha actin. Original objective magnification, × 32; T (tumor), L (Lung), **P<0.01, significant for V165, respectively, vs cV. (C and D) RT-Q-PCR analyses of VEGF isoforms, MMP-1 (C) and TNC (D) in isolated cVM, V189M and V165M lung tumours cells. TBP was used as an internal control. The relative mRNA levels of each gene in the control are set to 1, and values are expressed as means±s.e.m. *P<0.05, **P< 0.01. Scale bar, 250 μm.
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fig3: Stromal compartment and migration of gene expression. (A and B) Immunostaining (A) and quantification (B) for SM-alpha actin. For each animal, two sections per lung positive for tumour cells were immunostained with SM-alpha actin. Original objective magnification, × 32; T (tumor), L (Lung), **P<0.01, significant for V165, respectively, vs cV. (C and D) RT-Q-PCR analyses of VEGF isoforms, MMP-1 (C) and TNC (D) in isolated cVM, V189M and V165M lung tumours cells. TBP was used as an internal control. The relative mRNA levels of each gene in the control are set to 1, and values are expressed as means±s.e.m. *P<0.05, **P< 0.01. Scale bar, 250 μm.

Mentions: As the presence of SM-alpha actin-positive cells is strongly associated with tumour aggressiveness (Orimo et al, 2005), we performed immunostaining with the SM-alpha actin antibody to detect myofibroblasts in the compartment of lungs positive for V165-B and V189-B tumour cells (Figure 3A). The percentage of positive cells was higher in the lungs colonised by V165-B cells than in ones colonised by V189-B or cV-B cells (Figure 3A). The overexpression of VEGF165 and VEGF189 in isolated lungs positive for tumour cells (called V165, V189 and cV, respectively) was confirmed by RT–PCR (Figure 3C). The correlation between the expression evaluation by RT–PCR of the VEGF isoforms and their protein levels was previously demonstrated (Hervé et al, 2008). Besides, V189 cells also expressed significantly lower levels of MMP1mRNA than V165 cells (Figure 3C). We further investigated the expression in those clones of tenascin-C (TNC), a well-known marker of stroma associated with epithelial malignancy (Mackie et al, 1987; Dandachi et al, 2001). V189 cells expressed significantly lower levels of TNC mRNA than cV and V165 cells (Figure 3D).


MDA-MB-231 breast cancer cells overexpressing single VEGF isoforms display distinct colonisation characteristics
Stromal compartment and migration of gene expression. (A and B) Immunostaining (A) and quantification (B) for SM-alpha actin. For each animal, two sections per lung positive for tumour cells were immunostained with SM-alpha actin. Original objective magnification, × 32; T (tumor), L (Lung), **P<0.01, significant for V165, respectively, vs cV. (C and D) RT-Q-PCR analyses of VEGF isoforms, MMP-1 (C) and TNC (D) in isolated cVM, V189M and V165M lung tumours cells. TBP was used as an internal control. The relative mRNA levels of each gene in the control are set to 1, and values are expressed as means±s.e.m. *P<0.05, **P< 0.01. Scale bar, 250 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559830&req=5

fig3: Stromal compartment and migration of gene expression. (A and B) Immunostaining (A) and quantification (B) for SM-alpha actin. For each animal, two sections per lung positive for tumour cells were immunostained with SM-alpha actin. Original objective magnification, × 32; T (tumor), L (Lung), **P<0.01, significant for V165, respectively, vs cV. (C and D) RT-Q-PCR analyses of VEGF isoforms, MMP-1 (C) and TNC (D) in isolated cVM, V189M and V165M lung tumours cells. TBP was used as an internal control. The relative mRNA levels of each gene in the control are set to 1, and values are expressed as means±s.e.m. *P<0.05, **P< 0.01. Scale bar, 250 μm.
Mentions: As the presence of SM-alpha actin-positive cells is strongly associated with tumour aggressiveness (Orimo et al, 2005), we performed immunostaining with the SM-alpha actin antibody to detect myofibroblasts in the compartment of lungs positive for V165-B and V189-B tumour cells (Figure 3A). The percentage of positive cells was higher in the lungs colonised by V165-B cells than in ones colonised by V189-B or cV-B cells (Figure 3A). The overexpression of VEGF165 and VEGF189 in isolated lungs positive for tumour cells (called V165, V189 and cV, respectively) was confirmed by RT–PCR (Figure 3C). The correlation between the expression evaluation by RT–PCR of the VEGF isoforms and their protein levels was previously demonstrated (Hervé et al, 2008). Besides, V189 cells also expressed significantly lower levels of MMP1mRNA than V165 cells (Figure 3C). We further investigated the expression in those clones of tenascin-C (TNC), a well-known marker of stroma associated with epithelial malignancy (Mackie et al, 1987; Dandachi et al, 2001). V189 cells expressed significantly lower levels of TNC mRNA than cV and V165 cells (Figure 3D).

View Article: PubMed Central - PubMed

ABSTRACT

Background:: Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that has important roles in angiogenesis. Our knowledge of the significance of VEGF isoforms in human cancer remains incomplete.

Methods:: Bioluminescence imaging and transcriptomic analysis were used to study the colonisation capacity of the human breast cancer cells MDA-MB-231 controlling or overexpressing the VEGF165 or VEGF189 isoform (named cV-B, V165-B and V189-B, respectively) in nude mice.

Results:: When injected into the bloodstream, V189-B cells induced less metastasis in the lungs and bone than V165-B and cV-B control cells, consistent with longer survival of these mice and delay in tumour uptake in the mice injected with a V189-B clone. Histological analysis confirmed that there were less &alpha;SMA-positive cells in the lungs of the mice injected with V189-B. In vitro V189-B cells decreased both cell invasion and survival. Using transcriptomic analysis, we identified a subset of 18 genes expressed differentially between V189 and V165 cell lines and in 120 human breast tumours. V165 was associated with poor prognosis, whereas V189 was not, suggesting a complex regulation by VEGF isoforms. Our results showed a negative correlation between the expression pattern of VEGF189 and the levels of expression of seven genes that influence metastasis.

Conclusion:: Our findings provide the first evidence that VEGF isoforms have different effects on breast cancer cell line colonisation in vivo.

No MeSH data available.


Related in: MedlinePlus