Label‐free subcellular mapping of iron‐bound proteins in breast cancer cells

Abstract

Dysregulation of iron transport, storage and utilization is at the basis of a variety of human diseases, since free iron can participate in redox reactions that are deleterious to lipid membranes and signaling pathways. As intracellular iron transport occurs in a pH-dependent manner, changes in endosomal pH, often associated with cancer, could lead to alterations in iron homeostasis. Essential to iron transport is transferrin (Tf), which binds iron at a neutral pH for safe transport throughout the body and releases it upon uptake and delivery to a mildly acidified endosome. Excess iron can be stored in cytoplasmic protein ferritin (Ft) or delivered into mitochondria to be incorporated into proteins with heme or iron-sulfur structures. Recently, we have developed a novel label-free Raman hyperspectral microscopy methodology to identify the subcellular distributions and relative quantities of iron-bound Tf, Ft and heme proteins within heterogenous cell populations. Previously, we have shown that Raman hyperspectral imaging could identify endocytic distributions of iron-bound Tf using a specific Raman peak at 1300 cm-1 in intact breast cancer cells. Moreover, we have shown that alterations in the subcellular localization of iron-bound Tf populations correlated with altered endocytic regulation of iron-bound Tf in two distinct breast cancer cell lines. Based on these results, we propose that breast cancer cells display a disrupted iron homeostasis due to iron release delays caused by alterations in the pH or ionic milieu of the early endosomes. Currently, we are testing a putative correlation between pH gradient alterations and iron release delays in different breast cancer cells. To further investigate cellular iron regulation, Ft cell-specific Raman signature was determined via evaluation of cells incubated in the presence or absence of iron-loaded Ft. Since cells may internalize Ft through an endocytic pathway, this approach allowed for identification of Ft Raman signature. However, the subcellular distribution of internalized iron-loaded Ft may not be reflective of normal cytoplasmic Ft in cells. Internalization of iron-loaded Ft results in multiple significantly different Raman spectra peaks at several wavelengths. Importantly, we have used multivariate analysis to show a significant separation of cell Raman spectra from that of cells incubated with Ft. Further we can map a previously known heme protein peak at 1372 cm-1, which is specifically associated with the ferric iron heme. In the future, following further validation of Ft peaks the mapping of Tf, Ft and heme can be evaluated across multiple cell lines and tissue samples. The application of this research has far reaching potential in understanding how dysregulation of endocytic pH may lead to alterations in intracellular iron transport and storage in breast cancer cells.