Ruimin Huang

Surface-Enhanced Resonance Raman Scattering Nanostars for High Precision Cancer Imaging

Surface-enhanced resonance Raman scattering gold nanostars allow detection of macro- and microscopic foci of premalignant and cancerous lesions in vivo. Seeing Nanostars Microscopic tumors may be difficult for the naked eye to see, but they are no match for nanosized imaging agents, which home in on cancerous tissues to signal the presence of disease. Harmsen and colleagues created a new generation of cancer imaging agents, called “surface-enhanced resonance Raman scattering (SERRS) nanostars” −75-nm star-shaped gold cores wrapped in Raman reporter molecule-containing silica. When hit by a near-infrared laser, these nanostars emit a unique photonic signature (Raman “fingerprint”). The authors used a new silica encapsulation method and a reporter molecule that was “in resonance” with the laser, which meant that they shone nearly 400 times brighter than their “nonresonant” counterparts during Raman imaging. The SERRS nanostars were used to image macro- and microscopic malignant lesions in animal models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma with high precision. As endoscopic and handheld Raman imaging devices are further developed for the clinic, the SERRS nanostars are sure to find a place in human tumor detection. The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread, and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the true extent of tumors is desired clinically and surgically. We show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal locoregional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed SERRS nanostars. The SERRS nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In genetically engineered mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, and in one human sarcoma xenograft model, SERRS nanostars enabled accurate detection of macroscopic malignant lesions, as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 fM limit of detection) of SERRS nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS nanostars a promising imaging agent for more precise cancer imaging and resection.

Non-invasive mapping of deep-tissue lymph nodes in live animals using a multimodal PET/MRI nanoparticle

The invasion status of tumour-draining lymph nodes (LNs) is a critical indicator of cancer stage and is important for treatment planning. Clinicians currently use planar scintigraphy and single-photon emission computed tomography (SPECT) with (99m)Tc-radiocolloid to guide biopsy and resection of LNs. However, emerging multimodality approaches such as positron emission tomography combined with magnetic resonance imaging (PET/MRI) detect sites of disease with higher sensitivity and accuracy. Here we present a multimodal nanoparticle, (89)Zr-ferumoxytol, for the enhanced detection of LNs with PET/MRI. For genuine translational potential, we leverage a clinical iron oxide formulation, altered with minimal modification for radiolabelling. Axillary drainage in naive mice and from healthy and tumour-bearing prostates was investigated. We demonstrate that (89)Zr-ferumoxytol can be used for high-resolution tomographic studies of lymphatic drainage in preclinical disease models. This nanoparticle platform has significant translational potential to improve preoperative planning for nodal resection and tumour staging.

Guiding brain tumor resection using surface-enhanced Raman scattering nanoparticles and a hand-held Raman scanner.

The current difficulty in visualizing the true extent of malignant brain tumors during surgical resection represents one of the major reasons for the poor prognosis of brain tumor patients. Here, we evaluated the ability of a hand-held Raman scanner, guided by surface-enhanced Raman scattering (SERS) nanoparticles, to identify the microscopic tumor extent in a genetically engineered RCAS/tv-a glioblastoma mouse model. In a simulated intraoperative scenario, we tested both a static Raman imaging device and a mobile, hand-held Raman scanner. We show that SERS image-guided resection is more accurate than resection using white light visualization alone. Both methods complemented each other, and correlation with histology showed that SERS nanoparticles accurately outlined the extent of the tumors. Importantly, the hand-held Raman probe not only allowed near real-time scanning, but also detected additional microscopic foci of cancer in the resection bed that were not seen on static SERS images and would otherwise have been missed. This technology has a strong potential for clinical translation because it uses inert gold–silica SERS nanoparticles and a hand-held Raman scanner that can guide brain tumor resection in the operating room.

A long noncoding RNA Sox2ot regulates lung cancer cell proliferation and is a prognostic indicator of poor survival.

Sox2 overlapping transcript (Sox2ot) is a long noncoding RNA (lncRNA), localized on human chromosome 3q26.33, which is frequently amplified in lung squamous cell carcinomas (SCCs). However, its roles in lung cancer remain under investigation. In this study, we found that Sox2ot was up-regulated over two folds in 53.01% of human primary lung cancers (44/83). The expression level of Sox2ot is significantly higher in SCCs than that in adenocarcinomas (ADCs) of the lung. Further study found high Sox2ot expression predicted poor survival in lung cancer patients (P=0.0053), implying Sox2ot is a novel prognostic factor. In two human lung cancer cell lines, HCC827 and SK-MES-1, knocking down Sox2ot inhibited cell proliferation by inducing G2/M arrest, with a concomitant decrease of cells in S phase. Reduced protein levels of Cyclin B1 and Cdc2 were also observed. Importantly, knocking down Sox2ot decreased EZH2 expression and reintroduction of EZH2 allowed Sox2ot knockdown cells progressed through G2/M phase, which correlates with the restoration of Cyclin B1 and Cdc2 expressions. Altogether, our data suggested that Sox2ot plays an important role in regulating lung cancer cell proliferation, and may represent a novel prognostic indicator for the disease.

TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT

Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. However, it is unclear whether CAFs from UBC induce cell invasion and which signaling pathway is involved. Herein, we found that conditional medium from UBC CAFs (CAF-CM) enhanced the invasion of UBC cells. CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells. Higher concentration of TGFβ1 in CAF-CM, comparing with the CM from adjacent normal fibroblast, led to phosphorylation of Smad2 in UBC cells. Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion. Interestingly, a long non-coding RNA, ZEB2NAT, was demonstrated to be essential for this TGFβ1-dependent process. ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level. Consistently, TGFβ1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens. Our data revealed a novel mechanism that CAFs induces EMT and invasion of human UBC cells through the TGFβ1-ZEB2NAT-ZEB2 axis.

MYCN and MYC regulate tumor proliferation and tumorigenesis directly through BMI1 in human neuroblastomas.

The BMI1 gene is overexpressed in ~90% of human neuroblastomas. However, little is known about the regulation of BMI1 expression. Using microarray and immunohistochemical analysis, we show that BMI1 expression correlated with MYCN levels in MYCN‐amplified human neuroblastomas, and with MYC levels in the MYCN‐nonamplified group. We further demonstrated that BMI1 is a direct target gene of MYCN/MYC in 3 neuroblastoma cell lines: BE (2)‐C, LAN1, and SH‐SY5Y. Overexpression of MYCN or MYC transactivated the BMI1 promoter and up‐regulated BMI1 gene expression. shRNA‐mediated knockdown of MYCN or MYC decreased BMI1 gene expression. Chromatin immunoprecipitation and point‐mutation assays revealed that both MYCN and MYC bind to the E‐box within the BMI1 promoter. Overexpression of BMI1, MYCN, and MYC independently increased both cell proliferation and tumor growth. Conversely, specific inhibition of BMI1, MYCN, and MYC decreased tumor cell proliferation and tumor growth. Interestingly, BMI1 suppression in MYCN/MYC‐overexpressing cells resulted in significantly greater inhibition compared to that in mock‐transduced and parental cells. Our results indicate that MYCN and MYC regulate BMI1 gene expression at the transcriptional level and that dysregulation of the BMI1 gene mediated by MYCN or MYC overexpression, confers increased cell proliferation during neuroblastoma genesis and tumor progression.—Huang, R., Cheung, N.‐K. V., Vider, J., Cheung, I. Y., Gerald, W. L., Tickoo, S. K., Holland, E. C., Blasberg, R. G. MYCN and MYC regulate tumor proliferation and tumorigenesis directly through BMI1 in human neuroblastomas. FASEB J. 25, 4138–4149 (2011). www.fasebj.org

Rational Design of a Chalcogenopyrylium-Based Surface-Enhanced Resonance Raman Scattering-Nanoprobe with Attomolar Sensitivity

High sensitivity and specificity are two desirable features in biomedical imaging. Raman imaging has surfaced as a promising optical modality that offers both. Here, we report the design and synthesis of a group of near infrared absorbing 2-thienyl-substituted chalcogenopyrylium dyes tailored to have high affinity for gold. When adsorbed onto gold nanoparticles, these dyes produce biocompatible SERRS-nanoprobes with attomolar limits of detection amenable to ultrasensitive in vivo multiplexed tumor and disease marker detection.

Positron Lymphography: Multimodal, High-Resolution, Dynamic Mapping and Resection of Lymph Nodes After Intradermal Injection of 18F-FDG

The lymphatic system plays a critical role in the maintenance of healthy tissues. Its function is an important indicator of the presence and extent of disease. In oncology, metastatic spread to local lymph nodes (LNs) is a strong predictor of poor outcome. Clinical methods for the visualization of LNs involve regional injection and tracking of 99mTc-sulfur colloid (99mTc-SC) along with absorbent dyes. Intraoperatively, these techniques suffer from the requirement of administration of multiple contrast media (99mTc-SC and isosulfan blue), unwieldy γ-probes, and a short effective surgical window for dyes. Preclinically, imaging of transport through the lymphatics is further hindered by the resolution of lymphoscintigraphy and SPECT. We investigated multimodal imaging in animal models using intradermal administration of 18F-FDG for combined diagnostic and intraoperative use. PET visualizes LNs with high sensitivity and resolution and low background. Cerenkov radiation (CR) from 18F-FDG was evaluated to optically guide surgical resection of LNs. Methods: Imaging of 18F-FDG uptake used PET and sensitive luminescent imaging equipment (for CR). Dynamic PET was performed in both sexes and multiple strains (NCr Nude, C57BL/6, and Nu/Nu) of mice. Biodistribution confirmed the uptake of 18F-FDG and was compared with that of 99mTc-SC. Verification of uptake and the ability to use 18F-FDG CR to guide nodal removal were confirmed histologically. Results: Intradermal injection of 18F-FDG clearly revealed lymphatic vessels and LNs by PET. Dynamic imaging revealed rapid and sustained labeling of these structures. Biodistribution of the radiotracer confirmed the active transport of radioglucose in the lymphatics to the local LNs and over time into the general circulation. 18F-FDG also enabled visualization of LNs through CR, even before surgically revealing the site, and guided LN resection. Conclusion: Intradermal 18F-FDG can enhance the preclinical investigation of the lymphatics through dynamic, high-resolution, and quantitative tomographic imaging. Clinically, combined PET/Cerenkov imaging has significant potential as a single-dose, dual-modality tracer for diagnostics (PET/CT) and guided resection of LNs (Cerenkov optical).

Crizotinib, a c-Met Inhibitor, Prevents Metastasis in a Metastatic Uveal Melanoma Model

Uveal melanoma is the most common primary intraocular malignant tumor in adults and half of the primary tumors will develop fatal metastatic disease to the liver and the lung. Crizotinib, an inhibitor of c-Met, anaplastic lymphoma kinase (ALK), and ROS1, inhibited the phosphorylation of the c-Met receptor but not of ALK or ROS1 in uveal melanoma cells and tumor tissue. Consequently, migration of uveal melanoma cells was suppressed in vitro at a concentration associated with the specific inhibition of c-Met phosphorylation. This effect on cell migration could be recapitulated with siRNA specific to c-Met but not to ALK or ROS1. Therefore, we developed a uveal melanoma metastatic mouse model with EGFP–luciferase-labeled uveal melanoma cells transplanted by retro-orbital injections to test the effect of crizotinib on metastasis. In this model, there was development of melanoma within the eye and also metastases to the liver and lung at 7 weeks after the initial transplantation. When mice were treated with crizotinib starting 1 week after the transplantation, we observed a significant reduction in the development of metastases as compared with untreated control sets. These results indicate that the inhibition of c-Met activity alone may be sufficient to strongly inhibit metastasis of uveal melanoma from forming, suggesting crizotinib as a potential adjuvant therapy for patients with primary uveal melanoma who are at high risk for the development of metastatic disease. Mol Cancer Ther; 12(12); 2817–26. ©2013 AACR.

Integrin αvβ3-Targeted IRDye 800CW Near-Infrared Imaging of Glioblastoma

Purpose: Integrin αvβ3 plays an important role in tumor angiogenesis, growth, and metastasis. We have tested a targeted probe to visualize integrin receptor expression in glioblastomas using near-infrared fluorescent (NIRF) imaging. Experimental design: A transgenic glioblastoma mouse model (RCAS-PDGF-driven/tv-a glioblastoma, which mimics the infiltrative growth pattern of human glioblastomas) and two human orthotopic glioblastoma models (U-87 MG with high integrin β3 expression and TS543 with low integrin β3 expression) were studied. An integrin-targeting NIRF probe, IRDye 800CW-cyclic-RGD peptide (IRDye 800CW-RGD), was tested by in vivo and ex vivo NIRF imaging. Results: We show that the IRDye 800CW-RGD peptide: (i) specifically binds to integrin receptors; (ii) is selectively localized to glioblastoma tissue with overexpressed integrin receptors and is retained over prolonged periods of time; (iii) is associated with minimal autofluorescence and photobleaching because of imaging at 800 nm; (iv) provides delineation of tumor tissue with high precision because of a high tumor-to-normal brain fluorescence ratio (79.7 ± 6.9, 31.2 ± 2.8, and 16.3 ± 1.3) in the U-87 MG, RCAS-PDGF, and TS543 models, respectively; P < 0.01); and (v) enables fluorescence-guided glioblastoma resection. Importantly, small foci of residual fluorescence were observed after resection was completed using white light imaging alone, and these fluorescent foci were shown to represent residual tumor tissue by histology. Conclusions: NIRF imaging with the IRDye 800CW-RGD probe provides a simple, rapid, low-cost, nonradioactive, and highly translatable approach for improved intraoperative glioblastoma visualization and resection. It also has the potential to serve as an imaging platform for noninvasive cancer detection and drug efficacy evaluation studies. Clin Cancer Res; 18(20); 5731–40. ©2012 AACR.