In this study, we show that VEGF correlated significantly with leptin and with the leptin:adiponectin ratio in normal human breast tissue in situ. No correlations were found in abdominal s.c. fat tissue. Co-culture of adipocytes and ER+ breast cancer cells induced alterations per se with increased extracellular levels of VEGF and leptin, and enhanced the effects of estradiol compared with either cell type cultured alone. In vitro, VEGF had a regulatory effect on leptin and not vice versa. A dietary addition of ground flaxseed decreased the in vivo extracellular breast levels of leptin and leptin:adiponectin ratio whereas VEGF levels were unaltered.
The cellular composition of the breast provides a unique microenvironment, contributing to several steps in the carcinogenic process [
28]. This environment consists of different cell types such as epithelial-, endothelial-, immune-, and stroma cells and adipocytes. All of these cell types contribute to biologically active secreted factors in the extracellular space. This microenvironment is difficult to reproduce in
in vitro systems and commonly used techniques such as immunohistochemistry of tissues may not reflect the extracellular components. Microdialysis is a minimally invasive
in vivo technique, which enables sampling of molecules from the extracellular space of many organs. This sampling will, however, not distinguish from which cell type the molecules originate. The microdialysis technique has been adapted to sample bioactive molecules present
in situ in both normal and malignant human breast tissue [
15‐
17,
20,
29,
30]. By using microdialysis, it has previously been shown that estradiol regulates VEGF in normal human breast tissue
in vivo and in breast cancer models [
16‐
19,
30,
31]. VEGF, being a potent angiogenic factor, plays a key role in normal vascular growth, alongside with its involvement in several pathological conditions such as cancer [
9]. In addition to estrogen, VEGF expression may also be regulated by several other factors including hypoxia, cytokines, and growth factors [
32,
33]. In mouse mammary cancer cells, it has been shown that leptin increased the expression of VEGF and its receptor VEGF-R2 [
8] suggesting that VEGF may be regulated by leptin. Our present data indeed support an interaction between leptin and VEGF in normal human breast tissue
in situ as extracellular VEGF correlated significantly with leptin. None of these correlations were detected in abdominal s.c. fat suggesting tissue specific events in the breast. In our
in vitro neutralizations experiments, inhibition of VEGF decreased extracellular leptin levels in a dose-dependent fashion whereas inhibition of leptin had no influence on VEGF levels. In contrast to previous animal studies, our data does not support the concept that VEGF is under direct regulation of leptin in human tissues. Although co-culture of two different cell types may not reflect the complex environment in the breast tissue, it offers a more physiologically relevant experimental model compared to single cell cultures. In our present study, co-culture per se increased the extracellular levels of leptin and VEGF compared with the different cell types cultured alone, which imply the importance of intercellular communication. Moreover, the effects of estradiol exposure seemed to be enhanced when the cells were co-cultured compared to monoculture. Co-culture of adipocytes and breast cancer cells resulted in a doubling of the leptin levels compared to adipocytes cultured alone whereas the levels were tripled in presence of estradiol. This highlights the importance of the cross talk between different cell-types for the biological function of a tissue. As all established so-called normal breast epithelial cell lines do not express the ER, we chose to use ER positive breast cancer cells to be able to elucidate the role of estrogen in these regulations [
26,
27].
Life style factors such as diet have been shown to attenuate the risk of breast cancer in different populations [
34,
35] and previous studies have shown that a dietary addition of flaxseed to premenopausal women tipped the breast microenvironment into angiogenesis inhibition and anti-inflammatory conditions [
15,
21]. Here we show that flaxseed affected leptin and adiponectin levels whereas VEGF levels were unaltered. Adiponectin levels increased in eight out of ten women and in two women the increase was nearly ten-fold. The duration of the diet addition of flaxseed ranged between 26 and 33 days depending on the individual differences in menstrual cycle length. The ten-fold increase in the two women may reflect an altered response to the diet rather than a longer duration of the flaxseed addition as these women added flaxseed for 29 and 30 days respectively. Although our in vitro data suggested a VEGF dependent alteration of leptin the diet modification showed that leptin levels were affected even in absence of an alteration of VEGF. This suggests that flaxseed may regulate leptin and adiponectin by direct mechanisms or that indirectly mechanisms other than VEGF may be involved. Alterations of the microenvironment into a pro-tumorigenic state are key events in the initiation, growth, and progression of cancers including breast cancer. Autopsy studies have revealed that up to 15 % of all women have
in situ cancer in their breast whereas only 1 % of women in the same age range are diagnosed with breast cancer [
36]. A pro-tumorigenic environment may allow these
in situ cancers to develop into clinical cancer disease. Our present data suggest that the breast microenvironment may be affected by relatively modest dietary modification and that flaxseed may induce an anti-tumorigenic microenvironment.
In summary, this study shows that extracellular VEGF exhibits a positive correlation with leptin in normal human breast tissue in vivo. The in vitro cell culture experiments imply that VEGF affects leptin expression and not vice versa. Co-culture with ER+ breast cancer cells and adipocytes induced profound changes of the extracellular environment per se and enhanced the effects of estradiol. Dietary addition of flaxseed resulted in decreased leptin and increased adiponectin levels in normal human breast tissue. There is a need to develop safer and less toxic breast cancer preventive strategies. Diet alterations could be one preventive breast cancer measure and the regulation of the breast microenvironment needs to be taken into account in future mechanistic studies of such interventions.