Int. junction proteins, which contribute to decreasing cancer cell collective migration. studies also revealed AuNPs JNJ-28312141 and PPTT inhibit cancer cell migration and invasion.12,18 However, the mechanism of how AuNPs treatments inhibit cancer cell migration remains largely unresolved. While the mechanism of nanoparticles on inhibiting the migration of single cells has been explored in the previous works, the mechanism regarding collective cell migration has rarely been studied. In collective cancer cell migration, a group of cancer cells migrate together, which might be a more efficient route for metastasis possibly due to a diverse cell population seeding other organs or the multicellular signal integration engaged.19 Collective cell migration has been widely observed in human cancers, especially in human epithelial cancers such as breast cancer and colon cancer.19, 20 It requires both the contractility of the cytoskeleton filaments and the active interactions of neighboring cells through the cell-cell junctions that connect the cytoskeleton of the neighboring cells.21 This process is highly dynamic and regulated by signal transduction through protein phosphorylation.22C24 Given their important roles, it is imperative to understand the signals evolved in the cytoskeleton filaments and cell-cell junctions shortly after AuNRs and PPTT stimulation for the rational design of effective strategies to inhibit cancer metastasis. In the current study, we hypothesized that the integrin-targeting AuNRs and PPTT treatment could affect the cytoskeleton and cell junctions, due to their interactions and connections as a network, to result in the inhibition of collective cancer cell migration (as shown in Scheme 1 in the Experimental section). To test this hypothesis, quantitative mass spectrometry (MS)-based phosphoproteomics was employed to examine the signaling pathways upon the stimulation of AuNRs and PPTT. A primary signaling pathway map has been constructed to display a large number of identified alterations. Furthermore, super-resolution microscopy imaging techniques were used to visualize the changes of key cytoskeletal and cell junction proteins. Both phosphoproteomics and super-resolution imaging results indicated possible functions of the AuNRs and PPTT in regulating and changing JNJ-28312141 the architecture of the cytoskeletal filaments and cell junctions, contributing to the inhibition of collective cancer cell migration. Open in a separate window 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. RESULTS AND DISCUSSION Gold Nanorods and NIR Light Attenuate the Migration and Invasion of Cancer Cells The preparation of integrin targeted AuNRs was stated in our previous work.18 Briefly, AuNRs with a size of 25 ( 3) 6 ( 2) nm (length width) and an aspect ratio of 4.2 (Figure S1A, transmission electron microscopy (TEM) image) were synthesized using the seedless growth method.25 Optimal heat-generating efficacy in PPTT with these AuNRs has been demonstrated previously.26 To remove the cytotoxic cetyltrimethylammonium bromide (CTAB), the as synthesized AuNRs were washed twice with D.I. water. Then, the AuNRs were functionalized with polyethylene glycol thiol (PEG) and ArgCGlyCAsp (RGD) peptides to increase the biocompatibility.27 and obtain integrin targeting,28 respectively. The surface conjugations were confirmed by the red-shift of the longitudinal surface plasmon resonance (SPR) band (Figure S1B) and surface charge changes of the AuNRs (Figure S1C), consistent with the previous reports.18 The binding of RGD peptide to the cell surface integrin could enhance the endocytosis of AuNRs.29 The internalization Rabbit Polyclonal to ADAMDEC1 of AuNRs within the cervical cancer cell line HeLa, JNJ-28312141 was observed.