Identification of Transferrin Receptor 1 (TfR1) Overexpressed in Lung Cancer Cells, and Internalization of Magnetic Au-CoFe2O4 Core-Shell Nanoparticles Functionalized with Its Ligand in a Cellular Model of Small Cell Lung Cancer (SCLC)

Abstract

Lung cancer is, currently, one of the main malignancies causing deaths worldwide. To date, early prognostic and diagnostic markers for small cell lung cancer (SCLC) have not been systematically and clearly identified, so most patients receive standard treatment. In the present study, we combine quantitative proteomics studies and the use of magnetic core-shell nanoparticles (mCSNP's), first to identify a marker for lung cancer, and second to functionalize the nanoparticles and their possible application for early and timely diagnosis of this and other types of cancer. In the present study, we used label-free mass spectrometry in combination with an ion-mobility approach to identify 220 proteins with increased abundance in small cell lung cancer (SCLC) cell lines. Our attention was focused on cell receptors for their potential application as mCSNP's targets; in this work, we report the overexpression of Transferrin Receptor (TfR1) protein, also known as Cluster of Differentiation 71 (CD71) up to a 30-fold increase with respect to the control cell. The kinetics of endocytosis, evaluated by a flow cytometry methodology based on fluorescence quantification, demonstrated that receptors were properly activated with the transferrin supported on the magnetic core-shell nanoparticles. Our results are important in obtaining essential information for monitoring the disease and/or choosing better treatments, and this finding will pave the way for future synthesis of nanoparticles including chemotherapeutic drugs for lung cancer treatments.

Keywords: cluster of differentiation 71 (CD71); label-free; mass spectrometry; nanoparticles; small cell lung cancer (SCLC); transferrin receptor (TfR1).

Scheme 1

Methodology for functionalization of mCSNPs adapted with some modifications from Springer Protocols [22].

Figure 1

Reactome Pathways. Differentially expressed proteins are involved in 212 biological pathways with FDR ≤ 0.05. The bar plot shows the top 50 biological pathways. FDR values were transformed to a negative base 10 logarithm, which means that larger values on the abscissa axis indicate better-represented pathways.

Figure 2

TfR1 (TFRC) proteins interactome. TfR1 can exert varied actions on important biological pathways within the cancer cells such as metabolism of RNA, translation initiation complex formation, immune system, cell cycle, clathrin-mediated endocytosis, vesicle-mediated transport, membrane trafficking, and clathrin-derived vesicle budding, mainly mediated by its direct interaction with dysregulated actin-related proteins and ubiquitous scaffolding proteins such as YWHAZ.

Figure 3

Expression analysis of TfR1. (A) Dynamic ranges of H69AR and MRC5 cells show the position of the TfR1 protein; in tumor cells (foreground), TfR1 is ranked at number 15, while in normal cells (background), it is ranked at number 725. The difference in abundance was ~33-fold change (H69AR/MRC5) (B.1) Western blot analysis, indicating that the transferrin receptor was highly up-regulated in the H69AR cell line, presenting a change ten times more compared with the normal lung cell line (B.2). (C.1) Flow cytometry plots and the graphs of the fluorescence indicating a TfR1/TFRC signal (B−+) in a 90% gated (C.2). (B−: double negative signal, B−+: single positive signal (PE), B++: double positive signal (PE and AF488), B+−: single positive signal (AF488)). (C.3) Fluorescence intensity graph, in MRC5 and H69R cells.

Figure 4

TfR1 is present to a greater extent throughout the plasmatic membrane of H69AR (B) than in MRC5 (A) cell line. Indirect immunofluorescence image of TfR1 protein labeled with anti CD71 as a primary antibody, 1:500 incubated overnight and the secondary goat anti-mouse-FITC, 1:750 was incubated for 1 h at room temperature.

Figure 5

TEM micrograph of Au-CoFe₂O₄ mCSNPs, exhibiting monodisperse NPs of size 5–10 nm.

Figure 6

(A) HAADF imaging, Au-CoFe₂O₄ TX-100 0.1 M CTAB 0.1 M. (B) Core-shell scan mapping revealing a Core-shell nanoparticle, Au as a shell and Co-Fe as a Core.

Figure 7

1 Flow cytometry gatings for measuring the endocytic internalization in normal and SCLC cells. MRC5 and H69AR cells were incubated with Tf-AF488 (Tf) conjugated and CSNPs-Tf-AF488 conjugated (CSNP-Tf). Tf-AF488 was used as a positive control to measure the maximum fluorescence value of endocytosis in both cell lines. Cells were incubated at 37 °C to allow internalization of Tf-AF488 and the CSNP complex at t = 0.5, 8, and 16 h. (a.1) Cells were first gated by side scatter versus forward scatter in a blank sample. (a.2) Representative profiles of t = 0.5 h and t = 16 h of internalization of the CSNP complex in a control cell line (MRC5); we observed that fluorescence (endocytosis level) is the same in blanks and t = 0.5 and 16 h. (b.1) Cells were first gated by side scatter versus forward scatter in a blank sample. (b.2) Representative profiles of t = 0.5 h and t = 16 h of the CSNP complex in the SCLC cell line (H69AR), we observed that fluorescence (endocytosis level) is different t = 0.5 and t =16 h, in (b.3) the graph shows the change of the gated percentage of fluorescence of Tf-AF488 (black bars) and the CSNP complex (gray bars) versus time, the expression of the data as the means ± SEM.