Imaging of Endocytic Trafficking and Extracellular Vesicles Released Under Neratinib Treatment in ERBB2+ Breast Cancer Cells

Abstract

Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration-approved pan-ERBB inhibitor employed for the treatment of ERBB2⁺ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30-100 nm) ERBB2^- EVs and large (>100 nm) ERBB2⁺ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2⁺ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs.

Keywords: HER2; adjuvant therapy; breast cancer; disease-free survival; electron microscopy; endocytosis; extracellular vesicles; multivesicular bodies; neratinib; tyrosine kinase inhibitors.

Figure 1.

NE modulates ERBB2 trafficking in SKBR-3 cells. (A) Representative confocal microscopy images showing SKBR-3 treated for 72 hr with DMSO or 6-nM NE stained with antibodies against the ERBB2 extracellular domain (green signal), EEA1 (red signal), and CD63 (red signal). Nuclei were stained with DAPI (blue signal). Colocalization is shown as a merged yellow signal. Insets show signal colocalization at a higher magnification. Scale bars: 20 μm. (B) Cell lysates from SKBR-3 cells treated with DMSO or 6 nM for 2, 6, 24, and 72 hr and subjected to immunoblot analysis with antibodies against ERBB2. Vinculin was used as loading control. On the left side of the panel, the migration of protein molecular mass standards expressed in kDa is shown. The blot shown is representative of three independent experiments. Abbreviations: NE, neratinib; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2.

(A) Electron micrograph showing the cell surface of SKBR-3 cells. Black arrowheads point to clathrin-coated pits (CCPs) and clathrin-coated vesicles (CCVs). (B) Morphometry of CCP/Vs. Plasma membranes of 10 randomly chosen SKBR-3 cells were screened to count CCPs and CCVs. We considered as plasma membrane-derived CCVs those vesicles detected at the cell surface and within 200 nm from it. Data are expressed as mean ± SEM. Unpaired t-test, *p=0.028. Scale bar: 100 nm. (C) Representative immunofluorescence images of SKBR-3 cells treated with NE or DMSO and then incubated with 10-µg/ml Alexa488 Ac-LDL for 2 hr at 37C. Cells were then fixed and costained with DAPI to detect nuclei. Scale bar: 10 μm. (D). Graphs showing quantification analysis of Ac-LDL dots intensity in NE- versus DMSO-treated cells. Data are expressed as mean ± SEM from two independent experiments in which 50 cells were analyzed for each condition. Unpaired t-test, ****p<0.0001. (E) Representative immunofluorescence images of MFC7 cells treated with NE or DMSO and then incubated with 10-µg/ml Alexa488 Ac-LDL for 2 hr at 37C. Cells were then fixed and costained with DAPI to detect nuclei. Scale bar: 10 μm. (F). Graphs showing quantification analysis of Ac-LDL mean dots intensity and number of spots/cells in NE- versus DMSO-treated cells. Data are expressed as mean ± SEM from two independent experiments in which 100 cells were analyzed for each condition. Unpaired t-test, not significant. Abbreviations: SEM, standard error of the mean; NE, neratinib; Ac-LDL, acetylated LDL; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3.

(A) Left, representative confocal images of SKBR-3 cells treated with DMSO or NE and labeled for EEA1 (upper panel, red signal), CD63 (lower panel, red signal), and ERBB2 (green signal). Nuclei were stained with DAPI (blue signal). Scale bar: 20 μm. Insets show signal colocalization at a higher magnification. Upper right, quantification of the number and size of EEA1⁺ dots and per cell in NE- versus DMSO-treated cells are depicted and expressed as mean ± SEM from n=3 experiments; 50 cells were analyzed for each experiment. Insets show signal colocalization at a higher magnification. Lower right, quantification of the number and size of CD63⁺ dots and per cell in NE- versus DMSO-treated cells are depicted and expressed as mean ± SEM from n=3, 50 cells were analyzed for each experiment. **p=0.038. (B) Left, ultrastructural analysis of multivesicular bodies (MVBs) (arrowheads) in SKBR-3 cells treated with DMSO or NE. After 72 hr of treatment, SKBR-3 cells were further probed with BSA 5-nm gold for 2 hr at 37C and processed for flat embedding in epoxy resin. Ultrathin sections show BSA-gold labeling (black dots) in MVBs in DMSO- and NE-treated cells. Right, quantification showing a significant increase in the number of MVBs in NE-treated cells estimated over 30 randomly chosen cell profiles. Scale bars: 200 nm. Abbreviations: NE, neratinib; DAPI, 4′,6-diamidino-2-phenylindole; SEM, standard error of the mean.

Figure 4.

(A) Representative TEM images of immuno-labeled EVs indicating the presence of both ERBB2⁻ small EVs (arrows) and ERBB2⁺ larger EVs (arrowheads). Scale bar: 200 nm. (B) Morphometric analysis of EV number. Results are plotted as mean ± SEM from N=3 independent preparations. Unpaired t-test, ****p<0.0001. (C) TEM analysis of EV size. Data are visualized as box plots showing EV size of ERBB2⁻ versus ERBB2⁺ EVs. The plot shows the median value as a horizontal bar, and the interquartile range as a box; the whiskers are the minimum and maximum values. EVs were counted and measured on 30 randomly taken micrographs for each condition from n=3 independent experiments. Abbreviations: EV, extracellular vesicle; TEM, transmission electron microscopy; SEM, standard error of the mean; NE, neratinib.

Figure 5.

(A) High magnification images of EVs isolated from DMSO- and NE-treated SKBR-3 cells immuno-labeled for ERBB2 (9G6 antibody, 10-nm protein A gold), indicating the presence of ERBB2 (arrowheads) on EVs. Scale bar: 100 nm. (B) Morphometric TEM analysis showing that the total number of gold particles associated with EVs is significantly decreased in NE-treated cells. ERBB2 gold particles detected on EVs were counted on 30 randomly taken micrographs for each condition (n=3 independent experiments). Unpaired t-test, *p=0.011. Abbreviations: EV, extracellular vesicle; NE, neratinib; TEM, transmission electron microscopy.