Preventing Metastasis Using Gold Nanorod-Assisted Plasmonic Photothermal Therapy in Xenograft Mice.

Despite significant research regarding metastasis, there has been limited success in preventing it. However, gold nanoparticle (AuNP) technology has shown the potential to inhibit metastasis. Our earlier studies of gold nanorod-assisted plasmonic photothermal therapy (AuNRs-PPTT), where gold nanorods (AuNRs) were irradiated with near-infrared (NIR) light to induce heat, were utilized in slowing cancer cell migration in vitro. Herein, we have expanded the in vitro studies of the AuNRs-PPTT to xenograft mice to inhibit metastasis of mammary gland tumors. The study duration was 32 days from 4T1 cancer cell injections in four treatment groups: control (PBS), NIR Only, AuNRs, and AuNRs + NIR. Multiple AuNRs-PPTT treatment sessions with intratumoral AuNRs injections were conducted every 7 days on average on the mice. Photoacoustic spectroscopy has been utilized to study the distribution and aggregation of AuNRs within the tumors and the drainage of particles to the sentinel right subiliac lymph node. The photoacoustic results revealed that the AuNRs' shapes are still stable regardless of their heterogeneous distributions inside the mammalian tumor and lymph nodes. Bioluminescence imaging was used to monitor metastasis using luciferin labeling techniques and has shown that AuNRs-PPTT inhibited metastasis completely within the first 21 days. Moreover, proteomics was run to determine the most pivotal inhibitory pathways: NETosis, cell growth, cell proliferation, inflammation, and extracellular matrix (ECM) degradation. These five mechanisms are interdependent within related networks, which synergistically explains the molecular mechanism of metastasis inhibition by AuNRs-PPTT. The current in vivo data ensures the viability of PPTT applications in inhibiting metastasis in humans.

Targeting cancer cell integrins using gold nanorods in photothermal therapy inhibits migration through affecting cytoskeletal proteins.

Metastasis is responsible for most cancer-related deaths, but the current clinical treatments are not effective. Recently, gold nanoparticles (AuNPs) were discovered to inhibit cancer cell migration and prevent metastasis. Rationally designed AuNPs could greatly benefit their antimigration property, but the molecular mechanisms need to be explored. Cytoskeletons are cell structural proteins that closely relate to migration, and surface receptor integrins play critical roles in controlling the organization of cytoskeletons. Herein, we developed a strategy to inhibit cancer cell migration by targeting integrins, using Arg-Gly-Asp (RGD) peptide-functionalized gold nanorods. To enhance the effect, AuNRs were further activated with 808-nm near-infrared (NIR) light to generate heat for photothermal therapy (PPTT), where the temperature was adjusted not to affect the cell viability/proliferation. Our results demonstrate changes in cell morphology, observed as cytoskeleton protrusions-i.e., lamellipodia and filopodia-were reduced after treatment. The Western blot analysis indicates the downstream effectors of integrin were attracted toward the antimigration direction. Proteomics results indicated broad perturbations in four signaling pathways, Rho GTPases, actin, microtubule, and kinases-related pathways, which are the downstream regulators of integrins. Due to the dominant role of integrins in controlling cytoskeleton, focal adhesion, actomyosin contraction, and actin and microtubule assembly have been disrupted by targeting integrins. PPTT further enhanced the remodeling of cytoskeletal proteins and decreased migration. In summary, the ability of targeting AuNRs to cancer cell integrins and the introduction of PPTT stimulated broad regulation on the cytoskeleton, which provides the evidence for a potential medical application for controlling cancer metastasis.

Efficacy, long-term toxicity, and mechanistic studies of gold nanorods photothermal therapy of cancer in xenograft mice.

Gold nanorods (AuNRs)-assisted plasmonic photothermal therapy (AuNRs-PPTT) is a promising strategy for combating cancer in which AuNRs absorb near-infrared light and convert it into heat, causing cell death mainly by apoptosis and/or necrosis. Developing a valid PPTT that induces cancer cell apoptosis and avoids necrosis in vivo and exploring its molecular mechanism of action is of great importance. Furthermore, assessment of the long-term fate of the AuNRs after treatment is critical for clinical use. We first optimized the size, surface modification [rifampicin (RF) conjugation], and concentration (2.5 nM) of AuNRs and the PPTT laser power (2 W/cm2) to achieve maximal induction of apoptosis. Second, we studied the potential mechanism of action of AuNRs-PPTT using quantitative proteomic analysis in mouse tumor tissues. Several death pathways were identified, mainly involving apoptosis and cell death by releasing neutrophil extracellular traps (NETs) (NETosis), which were more obvious upon PPTT using RF-conjugated AuNRs (AuNRs@RF) than with polyethylene glycol thiol-conjugated AuNRs. Cytochrome c and p53-related apoptosis mechanisms were identified as contributing to the enhanced effect of PPTT with AuNRs@RF. Furthermore, Pin1 and IL18-related signaling contributed to the observed perturbation of the NETosis pathway by PPTT with AuNRs@RF. Third, we report a 15-month toxicity study that showed no long-term toxicity of AuNRs in vivo. Together, these data demonstrate that our AuNRs-PPTT platform is effective and safe for cancer therapy in mouse models. These findings provide a strong framework for the translation of PPTT to the clinic.

Simultaneous Time-Dependent Surface-Enhanced Raman Spectroscopy, Metabolomics, and Proteomics Reveal Cancer Cell Death Mechanisms Associated with Gold Nanorod Photothermal Therapy.

In cancer plasmonic photothermal therapy (PPTT), plasmonic nanoparticles are used to convert light into localized heat, leading to cancer cell death. Among plasmonic nanoparticles, gold nanorods (AuNRs) with specific dimensions enabling them to absorb near-infrared laser light have been widely used. The detailed mechanism of PPTT therapy, however, still remains poorly understood. Typically, surface-enhanced Raman spectroscopy (SERS) has been used to detect time-dependent changes in the intensity of the vibration frequencies of molecules that appear or disappear during different cellular processes. A complete proven assignment of the molecular identity of these vibrations and their biological importance has not yet been accomplished. Mass spectrometry (MS) is a powerful technique that is able to accurately identify molecules in chemical mixtures by observing their m/z values and fragmentation patterns. Here, we complemented the study of changes in SERS spectra with MS-based metabolomics and proteomics to identify the chemical species responsible for the observed changes in SERS band intensities during PPTT. We observed an increase in intensity of the bands at around 1000, 1207, and 1580 cm-1, which were assigned in the literature to phenylalanine, albeit with dispute. Our metabolomics results showed increased levels of phenylalanine, its derivatives, and phenylalanine-containing peptides, providing evidence for more confidence in the SERS peak assignments. To better understand the mechanism of phenylalanine increase upon PPTT, we combined metabolomics and proteomics results through network analysis, which proved that phenylalanine metabolism was perturbed. Furthermore, several apoptosis pathways were activated via key proteins (e.g., HADHA and ACAT1), consistent with the proposed role of altered phenylalanine metabolism in inducing apoptosis. Our study shows that the integration of the SERS with MS-based metabolomics and proteomics can assist the assignment of signals in SERS spectra and further characterize the related molecular mechanisms of the cellular processes involved in PPTT.

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

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.