Heterogeneity of Transferrin receptor expression, distribution and transferrin uptake in breast cancer cells in 2D and 3D spheroid cultures

Abstract

Alterations in the expression of proteins involved in iron homeostasis have been detected in cancer cells, and their levels appear to correlate inversely with patient survival. Thus, elucidating the mechanisms underlying defective iron transport is crucial to understand the faulty iron homeostasis that is typical of cancer cells. Rapidly proliferating cancer cells show increased expression of proteins that support iron import, ex. transferrin receptor (TfR), as well as decreased levels of proteins involved in iron export, ex. ferroportin. TfR (CD71 or p90) delivers iron into cells via the binding of iron‐loaded transferrin (holo‐Tf) and subsequent endocytosis and recycling of TfR‐Tf complexes. Tf is a commonly used ligand for receptor mediated drug delivery and molecular imaging of breast tumors. However, TfR expression and Tf uptake tumor heterogeneity (Figure 1) is a crucial issue that limits our understanding of the role of defective iron transport in tumor progression as well as the use of Tf in targeted drug delivery and tumor imaging. Our long‐term goal is to develop novel imaging methodologies and biochemical approaches to establish, quantitate and modify TfR expression and Tf uptake heterogeneity using tumor xenografts and 3D cancer spheroids (Figure 1). Herein, novel approaches to modulate Tf uptake will be tested for their effect on the heterogeneity of Tf delivery across solid tumors. Previously, increasing the serum concentration of iron‐deleted Tf (apo‐Tf) has been shown to alter Tf saturation as well as the intracellular distribution and level of TfR expression in mice models of anemia, e.g. β‐Thalassemia. We currently hypothesize that increasing concentration of apo‐Tf will lead to a re‐distribution of TfR to the surface of the cells, as well as altered trafficking of iron in cancer cells. We will test the effect of apo‐Tf in TfR expression and distribution in human breast cancer 2D cell cultures, 3D cancer spheroids and tumor xenografts. Firstly, Tf uptake plate reader assays have shown that 24h incubation with apo‐Tf modulates Tf uptake differently in non‐cancerous breast epithelial cells (MCF10A) and in human breast cancer cells (MDA‐MB‐231 and T47D cells). Immunofluorescence imaging of non‐cancerous vs. breast cancer cells has shown that apo‐Tf treatment induces significant changes in the intracellular distribution of TfR and early endosomal marker EEA1. These results suggest that treatment with apo‐Tf acts via alteration of TfR‐Tf endocytic trafficking pathway. Similar experiments will be performed to determine how incubation with apo‐Tf affects TfR expression and Tf uptake heterogeneity in 3D cancer spheroids. Ability to potentially alter TfR trafficking in breast cancer cells is highly significant not only to alter iron homeostasis as a way to reduce cancer progression but also to optimize Tf‐mediated drug delivery approaches. Funding source: This work was supported by the National Institute of Health (NIH) R01 BRG CA207725.Support or Funding InformationFunding source: This work was supported by the National Institute of Health (NIH) R01 BRG CA207725.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.