Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells

Abstract

The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

Fig 1. Schematic representation of the hTfR-EGFP chimera.

TfR is a type II transmembrane glycoprotein found primarily as a homodimer consisting of identical monomers joined by two disulfide bonds. Each monomer (760 amino acids) consists of three major domains as follows: a large glycosylated extracellular C-terminal domain (amino acids 90–760) involved in ligand binding, a single-pass transmembrane domain (amino acids 62–89), and a short intracellular N-terminal domain (amino acids 1–61). The EGFP moiety is fused to the amino terminus of hTfR.

Fig 2. Construction and Expression of hTfR-EGFP in CHO cells.

(A) Sal I and BglII restriction enzyme digestion analysis. M: 1kb DNA ladder; lane 1: pEGFP-hTfR, lane 2: pGEM-T-hTfR, lane 3: pEGFP-C1. (B) Immunoblot analysis of hTfR expression in cells. Cell lysates were probed by mouse anti-human TfR mAb. Left: representative WB picture of 4 separate experiments was shown. Right: Densitometric analysis of hTfR levels of the western blots. P values were calculated on the basis of Dunnett-t test (NS, not significant). (C) EGFP expression in CHO cells was detected by FCM and fluorescence microscope (insert).

Fig 3. mAb/Tf was bound (4°C) and endocytosed (37°C) by CHO-hTfR cells.

(A) 2×10⁵ CHO-hTfR cells were incubated with anti-hTfR mAb for 1h followed by PE-conjugated donkey anti-mouse Ab staining for 30min at 4°C (upper). Half cells were incubated at 37°C for another 30 min and then rinsed by ice-cold medium 1 (Lower). (B) 2×10⁵ CHO-hTfR cells were incubated with Alexa-Tf (5μg per tube) at 4°C (upper) or 37°C for 1h. Then 37°C incubated cells were rinsed with ice-cold medium 1 (lower). Fluorescence was developed using Olympus fluorescence microscope (60×).

Fig 4. Validation of the hTfR-EGFP specificity.

(A) Confocal imaging (4°C) and (B) flow cytometry studies on CHO and CHOvec(hTfR^-) cells, CHO-hTfR (hTfR⁺) cells, HepG2 (hTfR⁺-wt) cells demonstrated the hTfR targeting of mAb and Tf. (B) Upper: representative FCM pictures were shown. Lower: The bar graph represented FCM analysis of the percentage of PE positive cells. Mean values ± standard deviation (SD), n = 3; P values were calculated on the basis of SPSS 17.0 statistical software (NS, not significant).

Fig 5. Dynamics of hTfR-EGFP mediated endocytosis in living cells.

CHO-hTfR cells expressing hTfR-EGFP were grown in glass chambers overnight. (A) Cells were cultured with anti-hTfR mAb for 1h at 4°C, and then the chamber was mounted onto the microscope stage. 2 min after the supplement of secondary antibody conjugated with PE to the chamber, image acquisition of PE and EGFP fluorescences was performed. Images were acquired continuously at 2-min interval for 40min on a microscope stage. Presented were the selected images taken during this period. Images were indicated by time points. PE and EGFP segregation emerged from 18 min. (B and C) Anti-TfR mAb (AMCA secondary labeling) binding cells were incubated for another 5min or 30min at 37°C to allow internalization. PE conjugated anti- mouse LAMP-1/ EEA-1 antibody was used to label intracellular lysosome and endosome to show the colocalization with mAb and hTfR-EGFP chimera at different time point. EEA-1 for 5min and LAMP-1 for 30min.

Fig 6. CHO-hTfR cells preferentially took up anti-hTfR mAb-conjugated nanoparticles.

The same amounts of CHO-hTfR and CHO cells (2×10⁵) were co-incubated with HPPS or HPPS-mAb at concentration of 312.5 nM at 37°C for 30 min. After twice washes, EGFP⁺DIR-BOA⁺ double positive cells were measured by flow cytometry using APC-Cy7 color substitution. (A) Representative FCM pictures were shown. (B) The bar graph represented FCM analysis of the percentage of DIR-BOA positive cells. Mean values ± standard deviation (SD), n = 4; P values were calculated on the basis of SPSS 17.0 statistical software.