In the study, we aimed to assess the effect of an acidic microenvironment of the secretion of proteins by the human MCF-7 breast cancer cell line. At baseline, this cell line secreted the chemokine IP-10, growth factors GDF-15, VEGF, EGF, G-CSF, FGF, as well as myoglobin. There was no secretion of a number of other pro- and anti-inflammatory cytokines, as well as chemokines, which is in keeping with other studies [6, 7].
When treating the cells with acidic medium of pH 3.2, there was a general reduction in protein secretion, possibly indicating that highly acidic pH compromises cellular integrity and function. Of note, G-CSF concentration was not significantly affected by acidic medium. This may be advantageous, as G-CSF acts in an autocrine loop to enhance EMT, migration, and invasion of breast cancer cells through activation of Stat3 , and also impairs the activity of neutrophils, macrophages, and fibroblasts . EGF, and FGF expression were also not significantly affected, which may offer some tumour cell protection in an acidic microenvironment, although VEGF expression was significantly reduced (p<0.01), which may impact tumour angiogenesis under low pH conditions.
In contrast, tumour cell function was largely preserved under mildly acidic conditions with the expression of myoglobin and GDF-15, which was significantly elevated in cells treated at pH 6.5 for 1 hr. Myoglobin is a key effector of oxygen storage and distribution, and its expression is induced in epithelial tumours in response to mitogenic stimuli, oxidative stress, and hypoxia . In our study, myoglobin was expressed at a moderate level in the baseline samples, and although expression decreased at most treatment points, it was significantly elevated when treated with medium of pH 6.5 for 1 hr. (p<0.0001). Myoglobin may assist to scavenge nitric oxide (NO) and reactive oxygen species (ROS), as well as binding oxygen to provide an adaptive advantage to MCF-7 tumour cells. GDF-15 is a pleomorphic cytokine. It can promote tumour growth arrest and apoptosis (including in MCF-7 cell lines), but may also promote tumour proliferation in malignant glioma cells . More importantly, GDF-15 has significant immunomodulatory functions including blocking lymphocyte recruitment to the TME, dendritic cell (DC) maturation, and antigen presentation, promoting trafficking of tumour-associated macrophages (TAMs) to the TME, and preventing tumour destruction by natural killer cells [10-12].
In conclusion, we successfully identified a number of analytes that are secreted by MCF-7 cells. Levels of specific analytes were significantly increased when tumour cells were treated with acidic media of pH 6.5. These may provide a selective advantage, ensuring survival of tumour cells in the acidic TME, as well as preventing tumour cell destruction by the immune system. Thus, the acidic tumour microenvironment promotes the evolution of tumour cells, and identifying and targeting these biomarkers could be of prognostic and therapeutic use.