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. 2015 Sep 25;465(3):338-43.
doi: 10.1016/j.bbrc.2015.07.142. Epub 2015 Aug 1.

Intracellular Distribution of TM4SF1 and Internalization of TM4SF1-antibody Complex in Vascular Endothelial Cells

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Free PMC article

Intracellular Distribution of TM4SF1 and Internalization of TM4SF1-antibody Complex in Vascular Endothelial Cells

Tracey E Sciuto et al. Biochem Biophys Res Commun. .
Free PMC article

Abstract

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.

Keywords: Antibody drug conjugate; Antibody internalization; Endothelial cells; Microtubules; TM4SF1.

Conflict of interest statement

Conflicts of interests

The authors have declared that no competing interests exist.

Figures

Fig. 1
Fig. 1
Distribution of TM4SF1 in HUVEC (A) and in human gastric cancer vascular endothelial cells (B) as determined by immuno-nanogold transmission electron microscopy. HUVEC cultured on glass discs and a resected gastric adenocarcinoma and immunostained with mouse anti-human TM4SF1 antibody 8G4, followed by nanogold labeled anti-mouse IgG1. (Aa,b) Nanogold deposits localized TM4SF1 in HUVEC to the plasma membrane (blue arrows), cytoplasmic vesicles (yellow circles), nuclear membrane and nuclear pores (red arrows), and the nucleoplasm (red circles). (Ac) Rough endoplasmic reticulum demonstrates extensive nanogold labeling. (Ba,b) TM4SF1 labeling of human gastric cancer vascular endothelial cells is similar to that in HUVEC. Luminal membrane staining (Ba) is much stronger than that of albuminal labeling (Bb, pink arrows). Scale bars, 100 nm.
Fig. 2
Fig. 2
Internalization of anti-TM4SF1 monoclonal antibody 8G4 in HUVEC. HUVEC were incubated with 8G4 or with control mouse-IgG1 (mIgG1) for 1h at 4°C, w ashed, and returned to culture at 37° C for varying periods of time to follow 8G4 uptake by (A) immunofluorescence microscopy, (B) immuno-nanogold transmission electron microscopy, and (C) immunoblotting. (A) 8G4 achieved maximal intensity in cytoplasm and nucleus at 4h and was largely lost at 24h. (B) At 4h, gold particles are demonstrated in the cytoplasm (yellow circles), associated with the nuclear membrane and nuclear pores (red arrows) and within the nucleoplasm (red circles). (C) Immunoblot of 8G4 pull down prepared 4h after returning HUVEC to culture demonstrates 28-kD form of TM4SF1.
Fig. 3
Fig. 3
Internalization of TM4SF1 in HUVEC in the presence of different inhibitors. HUVEC were pre-labeled with 8G4 at 4°C (A) and returned to culture at 37°C without (B) or in the presence of the following inhibitors: (C) 20 µM pitstop-2 (clathrin inhibitor), (D) 10 µM chloropromazine (clathrin and caveolin mediated endocytosis inhibitor), (E) 0.4 µM bifilomycin A (autophagy Inhibitor), or (F) 20 µM dynasore (dynamin inhibitor). After 4h, cells were fixed in 4% paraformaldehyde. Immunocytochemistry demonstrates substantial and equivalent 8G4 uptake at 4h with no added inhibitor or in the presence of pitstop-2, chloropromazine, or bifilomycin A. However, 8G4 remained largely on the cell surface when cultured with dynasore.
Fig. 4
Fig. 4
TM4SF1 internalization along microtubules. HUVEC pre-labeled with 8G4 at 4°C and returned to culture at 37°C were harvested at 4h for immunostaining. 8G 4 (green) and α-tubulin (red) immunofluorescence signals co-localized. (A) Z-stacked confocal image (27-stacks; 220 nm/stack) and a representative higher-resolution stack-8 image (inset i) show that internalized 8G4 is closely associated with α-tubulin stained microtubules. (B) Super-resolution Structured Illumination Microscopy demonstrate 8G4 signals in association with α-tubulin stained microtubules.

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