Selective Uptake and Intracellular Distribution of Covalently Multimerized Human Stem Cell Factor (SCF)-Fusion Proteins into Human c-kit+ Acute Myeloid Leukemia (AML) Cells.
Authors: Sadowski Julia , Erben Ulrike , Richter Jan F. , Bleiss Wilfrid , Bittroff-Leben Alexandra , Thiel Eckhard , Krauss Michael , Notter Michael 
Published: April 2020

Abstract
C-kit expression has been detected in primary blasts of human AML patients frequently at high levels, but to a much lower extent in normal hemopoietic progenitor cells. As previously shown, a trimeric SCF-immunoglobulin (Ig)G1 fusion protein induced more profound modulation of c-kit compared to native SCF (Erben et al Cancer Res59: 2924–2930, 1999). In this study we investigated the life span and distribution of recombinant multimeric SCF proteins intracellularly. Furthermore, we asked if c-kit modulation by SCF is useful to selectively target therapeutics into AML blasts. The CS-1 cell line established from a patient with monoblastic AML relapsing after allogeneic stem cell transplantation showed increasing c-kit expression over time in vivo and in vitro and was used as a model system for high-risk AML. Sorted CD34+ hemopoietic progenitor cells served as non-malignant control. In addition to SCF-IgG1, further recombinant SCF fusion proteins were generated by eukaryotic expression: SCF linked to an enhanced green fluorescent protein (SCF-EGFP reporter protein) and myc-tagged SCF dimerized via the hinge-region of human IgG1 (SCF-μ transporter protein). SCF-μ was bound to the surface of myc antibody-coated nanoparticles (SCF-μ/B model therapeutic carrier). Corresponding to initial surface c-kit expression levels, SCF-EGFP (10 nM) modulated c-kit on the total population of CS-1 AML blasts and on only a minor subfraction of non-malignant CD34+ progenitor cells with a maximum observed at 90 min as determined by flow cytometry. SCF-IgG1 fusion protein but not human IgG1 inhibited SCF-EGFP binding in a dose-dependent manner indicating receptor specificity. Confocal laser scanning microscopy revealed initial focal aggregation of SCF-EGFP localized to one cell pole of the surface of CS-1 followed by apparent internalization and intracellular redistribution represented by circumferential multiple microspots at 20 minutes following incubation. SCF-μ showed a comparable cellular distribution pattern over time. Intracellular staining signals increased in intensity when CS-1 cells were incubated with SCF-μ/B compared to free SCF-μ indicating increased delivery of SCF-μ when attached to nanoparticles. SCF-μ/B clearly co-localized with internalized red fluorescent transferrin within CD71+ CS-1 cells in co-incubation experiments consistent with their strong association with the endosomal compartment. Internalization and subcellular distribution was confirmed by transmission electron microscopy demonstrating nanoparticles being localized in both endosomes and cytoplasm. In summary, augmented c-kit modulation on leukemia cells by covalently dimerized SCF-μ freely or bound to nanoparticles is a new transport mechanism resulting in their intracellular delivery. Since nanoparticles can be loaded with bioactive compounds, e.g. toxins or small molecules, this approach may offer new options for targeted therapy of c-kithigh AML.