Gold Nanorod Photothermal Therapy Alters Cell Junctions and Actin Network in Inhibiting Cancer Cell Collective Migration

Abstract

Most cancer-related deaths come from metastasis. It was recently discovered that nanoparticles could inhibit cancer cell migration. Whereas most researchers focus on single-cell migration, the effect of nanoparticle treatment on collective cell migration has not been explored. Collective migration occurs commonly in many types of cancer metastasis, where a group of cancer cells move together, which requires the contractility of the cytoskeleton filaments and the connection of neighboring cells by the cell junction proteins. Here, we demonstrate that gold nanorods (AuNRs) and the introduction of near-infrared light could inhibit the cancer cell collective migration by altering the actin filaments and cell junctions with significantly triggered phosphorylation changes of essential proteins, using mass spectrometry-based phosphoproteomics. Further observation using super-resolution stochastic optical reconstruction microscopy (STORM) showed the actin cytoskeleton filament bundles were disturbed, which is difficult to differentiate under a normal fluorescence microscope. The decreased expression level of N-cadherin junctions and morphological changes of tight junction protein zonula occludens 2 were also observed. All of these results indicate possible functions of the AuNR treatments in regulating and remodeling the actin filaments and cell junction proteins, which contribute to decreasing cancer cell collective migration.

Keywords: STORM; collective cancer cell migration; gold nanorods; metastasis; phosphoproteomics; plasmonic photothermal therapy; super-resolution microscopy.

Figure 1.

Cellular uptake, cytotoxicity and motility upon AuNRs treatments. (A-B) Differential interference contrast (DIC) microscopic images of HeLa cells without (A) and with AuNRs@RGD after 24 h incubation (B). (C) DIC image of AuNRs@RGD distribute in the cell junction areas after 24 h incubation. The red arrows identify the locations of AuNRs. (D) Cell viability of HeLa cells after AuNRs and AuNRs+NIR treatments (n=3). (E) Western blotting for the BAX protein upon different treatments. (F and G) Scratch assay of HeLa cells (control, AuNRs treatment, and AuNRs+PPTT treatment) at 0 and 12 h (n=6). Student’s t test was used for statistical analysis. All values are expressed as means ± standard errors of the mean (SEM). ***p<0.001, **p< 0.01, *p<0.05. If not specified otherwise, “AuNRs” in all other figures means “AuNRs conjugated with RGD ligands”.

Figure 2.

Phosphoproteomics results. (A) Experimental workflow. Two comparisons were performed in data analysis. Comparison #1 (AuNRs vs. control): (B) Heatmap and (C) pathway analysis after AuNRs treatment. (D) Western blotting showing the altered phosphorylation sited in p120 Catenin (HeLa cells). (E) Altered phosphorylation sited in p120 Catenin (pS268) indicated by phosphoproteomics (HeLa cells). Comparison #2 (AuNRs + NIR vs. AuNRs): (F) Heatmap and (G) pathway analysis after AuNRs + NIR treatment. (H) Western blotting showing the altered phosphorylation sited in GSK3 (HeLa cells). (I) Altered phosphorylation sites GSK3 (pY216) indicated by phosphoproteomics (HeLa cells). Mean values in are shown in the heatmaps (n=3).

Figure 3.

Schematic diagram of the signaling pathways that are engaged with the cytoskeleton and cell junctions upon the AuNRs and PPTT treatment. The blue and red “P”s indicate the altered phosphorylation level upon AuNRs treatment and PPTT treatment (AuNRs+NIR), respectively.

Figure 4.

STORM and epifluorescence images of actin filaments in the cell-cell junction upon different treatments: (A, D) Control; (B, E) AuNRs; (C, F) AuNRs + NIR. After NIR exposure, the actin filaments at cell junctions exhibited clearly altered morphology (scale bar = 5 μm).

Figure 5.

(A-C) Immunofluorescence images of N-cadherin in HeLa cells before (A) and after AuNRs (B) and AuNRs+PPTT (C) treatments (more images in Figure S14). The fluorescence intensities in these images are normalized together. (D) The fluorescence quantification of the N-cadherin (n=20 cells, ±SEM). (E) Western blot results also indicate a decreased expression level of N-cadherin after treatments. (F) Immunofluorescence images of tight junction protein ZO-2 in MCF-7 cells, before and after AuNRs or AuNRs+PPTT treatments. The morphology of ZO-2 change from a normal and continuous line-like structure in the control group to a discontinuous dot-like structure after treatments. The figures showed 3D scanning of ZO-2, where Layer 1 is close to the bottom of the cells, and Layer 3 is close to the top of the cells. Scale bar = 20 μm.

Scheme 1.

Experimental design (A) and proposed mechanism (B) of AuNRs and PPTT in inhibiting cancer collective migration. Targeting integrin could affect the actin cytoskeleton and cell junctions to result in the inhibition of cancer cell collective migration. Phosphoproteomics and super-resolution fluorescence imaging, as well as Western blot, were the main experimental tools used in the current study.