Detection of Premalignant Gastrointestinal Lesions Using Surface-Enhanced Resonance Raman Scattering-Nanoparticle Endoscopy.

Cancers of the gastrointestinal (GI) tract are among the most frequent and most lethal cancers worldwide. An important reason for this high mortality is that early disease is typically asymptomatic, and patients often present with advanced, incurable disease. Even in high-risk patients who routinely undergo endoscopic screening, lesions can be missed due to their small size or subtle appearance. Thus, current imaging approaches lack the sensitivity and specificity to accurately detect incipient GI tract cancers. Here we report our finding that a single dose of a high-sensitivity surface-enhanced resonance Raman scattering nanoparticle (SERRS-NP) enables reliable detection of precancerous GI lesions in animal models that closely mimic disease development in humans. Some of these animal models have not been used previously to evaluate imaging probes for early cancer detection. The studies were performed using a commercial Raman imaging system, a newly developed mouse Raman endoscope, and finally a clinically applicable Raman endoscope for larger animal studies. We show that this SERRS-NP-based approach enables robust detection of small, premalignant lesions in animal models that faithfully recapitulate human esophageal, gastric, and colorectal tumorigenesis. This method holds promise for much earlier detection of GI cancers than currently possible and could lead therefore to marked reduction of morbidity and mortality of these tumor types.

High Precision Imaging of Microscopic Spread of Glioblastoma with a Targeted Ultrasensitive SERRS Molecular Imaging Probe.

The dismal prognosis of patients with malignant brain tumors such as glioblastoma multiforme (GBM) is attributed mostly to their diffuse growth pattern and early microscopic tumor spread to distant regions of the brain. Because the microscopic tumor foci cannot be visualized with current imaging modalities, it remains impossible to direct treatments optimally. Here we explored the ability of integrin-targeted surface-enhanced resonance Raman spectroscopy (SERRS) nanoparticles to depict the true tumor extent in a GBM mouse model that closely mimics the pathology in humans. The recently developed SERRS-nanoparticles have a sensitivity of detection in the femtomolar range. An RGD-peptide-conjugated version for integrin-targeting (RGD-SERRS) was compared directly to its non-targeted RAD-SERRS control in the same mice via Raman multiplexing. Pre-blocking with RGD peptide before injection of RGD-SERRS nanoparticles was used to verify the specificity of integrin-targeting. In contrast to the current belief that the enhanced permeability and retention (EPR) effect results in a baseline uptake of nanoparticles regardless of their surface chemistry, integrin-targeting was shown to be highly specific, with markedly lower accumulation after pre-blocking. While the non-targeted SERRS particles enabled delineation of the main tumor, the RGD-SERRS nanoparticles afforded a major improvement in visualization of the true extent and the diffuse margins of the main tumor. This included the detection of unexpected tumor areas distant to the main tumor, tracks of migrating cells of 2-3 cells in diameter, and even isolated distant tumor cell clusters of less than 5 cells. This Raman spectroscopy-based nanoparticle-imaging technology holds promise to allow high precision visualization of the true extent of malignant brain tumors.

Imaging of Liver Tumors Using Surface-Enhanced Raman Scattering Nanoparticles.

Complete surgical resection is the ideal first-line treatment for most liver malignancies. This goal would be facilitated by an intraoperative imaging method that enables more precise visualization of tumor margins and detection of otherwise invisible microscopic lesions. To this end, we synthesized silica-encapsulated surface-enhanced Raman scattering (SERS) nanoparticles (NPs) that act as a molecular imaging agent for liver malignancies. We hypothesized that, after intravenous administration, SERS NPs would avidly home to healthy liver tissue but not to intrahepatic malignancies. We tested these SERS NPs in genetically engineered mouse models of hepatocellular carcinoma and histiocytic sarcoma. After intravenous injection, liver tumors in both models were readily identifiable with Raman imaging. In addition, Raman imaging using SERS NPs enabled detection of microscopic lesions in liver and spleen. We compared the performance of SERS NPs to fluorescence imaging using indocyanine green (ICG). We found that SERS NPs delineate tumors more accurately and are less susceptible to photobleaching. Given the known advantages of SERS imaging, namely, high sensitivity and specific spectroscopic detection, these findings hold promise for improved resection of liver cancer.

Surface-enhanced resonance Raman scattering nanostars for high-precision cancer imaging.

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.

A "schizophotonic" all-in-one nanoparticle coating for multiplexed SE(R)RS biomedical imaging.

SERS nanoprobes for in vivo biomedical applications require high quantum yield, long circulation times, and maximum colloidal stability. Traditional synthetic routes require high metal-dye affinities and are challenged by unfavorable electrostatic interactions and limited scalability. We report the synthesis of a new near-IR active poly(N-(2-hydroxypropyl) methacrylamide) (pHPMA). The integration of various SERS reporters into a biocompatible polymeric surface coating allows for controlled dye incorporation, high colloidal stability, and optimized in vivo circulation times. This technique allows the synthesis of very small (<20 nm) SERS probes, which is crucial for the design of excretable and thus highly translatable imaging agents. Depending on their size, the "schizophotonic" nanoparticles can emit both SERS and fluorescence. We demonstrate the capability of this all-in-one gold surface coating and SERS reporter for multiplexed lymph-node imaging.

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.

Relationships between LDH-A, lactate, and metastases in 4T1 breast tumors.

PURPOSE: To investigate the relationship between lactate dehydrogenase A (LDH-A) expression, lactate concentration, cell metabolism, and metastases in murine 4T1 breast tumors. EXPERIMENTAL DESIGN: Inhibition of LDH-A expression and protein levels were achieved in a metastatic breast cancer cell line (4T1) using short hairpin RNA (shRNA) technology. The relationship between tumor LDH-A protein levels and lactate concentration (measured by magnetic resonance spectroscopic imaging, MRSI) and metastases was assessed. RESULTS: LDH-A knockdown cells (KD9) showed a significant reduction in LDH-A protein and LDH activity, less acid production, decreased transwell migration and invasion, lower proliferation, reduced glucose consumption and glycolysis, and increase in oxygen consumption, reactive oxygen species (ROS), and cellular ATP levels, compared with control (NC) cells cultured in 25 mmol/L glucose. In vivo studies showed lower lactate levels in KD9, KD5, and KD317 tumors than in NC or 4T1 wild-type tumors (P < 0.01), and a linear relationship between tumor LDH-A protein expression and lactate concentration. Metastases were delayed and primary tumor growth rate decreased. CONCLUSIONS: We show for the first time that LDH-A knockdown inhibited the formation of metastases, and was accompanied by in vivo changes in tumor cell metabolism. Lactate MRSI can be used as a surrogate to monitor targeted inhibition of LDH-A in a preclinical setting and provides a noninvasive imaging strategy to monitor LDH-A-targeted therapy. This imaging strategy can be translated to the clinic to identify and monitor patients who are at high risk of developing metastatic disease.