Angiogenesis and lymphangiogenesis are downregulated in primary breast cancer

Abstract

Background: Angiogenesis and lymphangiogenesis are considered to play key roles in tumour growth, progression and metastasis. However, targeting tumour angiogenesis in clinical trials showed only modest efficacy. We therefore scrutinised the concept of tumour angiogenesis and lymphangiogenesis by analysing the expression of crucial markers involved in these processes in primary breast cancer.

Methods: We analysed the expression of angiogenic, lymphangiogenic or antiangiogenic factors, their respective receptors and specific markers for endothelial and lymphendothelial cells by quantitative real-time RT-PCR in primary breast cancer and compared the expression profiles to non-cancerous, tumour-adjacent tissues and breast tissues from healthy women.

Results: We found decreased mRNA amounts of major angiogenic and lymphangiogenic factors in tumour compared to healthy tissues, whereas antiangiogenic factors were upregulated. Concomitantly, angiogenic and lymphangiogenic receptors were downregulated in breast tumours. This antiangiogenic, antilymphangiogenic microenvironment was even more pronounced in aggressive tumours and accompanied by reduced amounts of endothelial and lymphatic endothelial cell markers.

Conclusion: Primary breast tumours are not a site of highly active angiogenesis and lymphangiogenesis. Selection for tumour cells that survive with minimal vascular supply may account for this observation in clinical apparent tumours.

Figure 1

Expression of angiogenic, antiangiogenic and lymphangiogenic growth factors and chemokines in primary breast cancer. The mRNA expression of angiogenic, antiangiogenic and lymphangiogenic growth factors (A) and chemokines (B) was quantified by real-time RT-PCR in breast cancer tissues and tumour-adjacent tissues of the same patients. The ratio of the expression in tumour tissue to the respective tumour-adjacent tissue was calculated for each patient. The bars show the mean ratio with s.e.m. for 41 patients. ^*P<0.05, ^**P<0.01 and ^***P<0.001.

Figure 2

Comparison of expression levels of angiogenic, antiangiogenic and lymphangiogenic growth factors, and chemokines in primary breast cancer according to histopathologic grading. Relative expression data for the different growth factors and chemokines were analysed according to the histopathologic grading of the primary breast tumours. The horizontal line indicates the median of each group. ^*P<0.05.

Figure 3

Expression of receptors for angiogenic and lymphangiogenic factors in primary breast cancer. The mRNA expression of angiogenic and lymphangiogenic receptors was quantified by real-time RT-PCR in breast cancer tissues and tumour-adjacent tissues of the same patients. The ratio of the expression in tumour tissue to the respective tumour-adjacent tissue was calculated for each patient. The bars show the mean ratio with s.e.m. for 41 patients. ^*P<0.05 and ^***P<0.001.

Figure 4

Endothelial and lymphatic endothelial cells in primary breast cancer. (A) The mRNA expression of markers for endothelial cells (CD146, VE-Cadherin) and lymphatic endothelial cells (LYVE1, PROX1) was quantified by real-time RT-PCR in breast cancer tissues and tumour-adjacent tissues of the same patients. The ratio of the expression in tumour tissue to the respective tumour-adjacent tissue was calculated for each patient. The bars show the mean ratio with s.e.m. for 41 patients. ^***P<0.001. (B) Immunohistochemical detection of endothelial cells in different breast cancer samples and corresponding normal breast tissue. Tissue samples were stained for CD31 with DAB (brown) and counterstained with haematoxylin (blue). Magnification × 10.

Figure 5

Quantification of angiogenic and antiangiogenic growth factors in serum. The concentration of angiogenic and antiangiogenic factors were determined by ELISA in the sera of breast cancer patients at the time of diagnosis and the sera of healthy controls. The horizontal line indicates the median of each group. ^***P<0.001.