Daniel A. Orringer

Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering microscopy provides a rapid, label-free means of detecting tumor infiltration of brain tissue ex vivo and in vivo. Virtual Histology During brain tumor surgery, precision is key. Removing healthy tissue can cause neurologic deficits; leaving behind tumor tissue can allow cancer to spread and treatment to fail. To help the surgeon clearly see tumor versus normal tissue, Ji and colleagues developed a stimulated Raman scattering (SRS) microscopy method and demonstrated its ability to identify malignant human brain tissue. In SRS microscopy, laser beams are directed at the tissue sample to generate a series of output signals called “Raman spectra.” These spectra depend on the molecular composition of the tissue. Ji et al. implanted human brain cancer (glioblastoma) cells into mice, allowed them to infiltrate and grow into tumors, and then removed slices for SRS imaging. From the resulting spectra, the authors were able to differentiate the two major components of brain tissue—lipid-rich white matter and protein-rich cortex—as well as tumors, which are full of proteins. Intraoperatively, using an imaging window into mouse brains, the authors found that SRS microscopy could locate tumor infiltration in areas that appeared normal by eye, which suggests that this tool could be applied during surgery. Imaging fresh tissue slices ex vivo could also complement or perhaps replace standard hematoxylin and eosin (H&E) staining in the clinic because it avoids artifacts inherent in imaging frozen or fixed tissues. To this end, Ji and colleagues showed that SRS microscopy could identify hypercellular tumor regions in fresh surgical specimens from a patient with glioblastoma. Certain diagnostic features were present in these specimens and readily identified by SRS, including pseudopalisading necrosis and microvascular proliferation. The next step will be to apply SRS microscopy to a large collection of human specimens to see whether this technology may be useful in quickly distinguishing glioblastoma from healthy tissue, both outside and inside the operating room. Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

Ambient mass spectrometry for the intraoperative molecular diagnosis of human brain tumors

The main goal of brain tumor surgery is to maximize tumor resection while preserving brain function. However, existing imaging and surgical techniques do not offer the molecular information needed to delineate tumor boundaries. We have developed a system to rapidly analyze and classify brain tumors based on lipid information acquired by desorption electrospray ionization mass spectrometry (DESI-MS). In this study, a classifier was built to discriminate gliomas and meningiomas based on 36 glioma and 19 meningioma samples. The classifier was tested and results were validated for intraoperative use by analyzing and diagnosing tissue sections from 32 surgical specimens obtained from five research subjects who underwent brain tumor resection. The samples analyzed included oligodendroglioma, astrocytoma, and meningioma tumors of different histological grades and tumor cell concentrations. The molecular diagnosis derived from mass-spectrometry imaging corresponded to histopathology diagnosis with very few exceptions. Our work demonstrates that DESI-MS technology has the potential to identify the histology type of brain tumors. It provides information on glioma grade and, most importantly, may help define tumor margins by measuring the tumor cell concentration in a specimen. Results for stereotactically registered samples were correlated to preoperative MRI through neuronavigation, and visualized over segmented 3D MRI tumor volume reconstruction. Our findings demonstrate the potential of ambient mass spectrometry to guide brain tumor surgery by providing rapid diagnosis, and tumor margin assessment in near–real time.

Intraoperative mass spectrometry mapping of an onco-metabolite to guide brain tumor surgery

Significance The diagnosis of tumors during surgery still relies principally on an approach developed over 150 y ago: frozen section microscopy. We show that a validated molecular marker—2-hydroxyglutarate generated from isocitrate dehydrogenase 1 mutant gliomas—can be rapidly detected from tumors using a form of ambient MS that does not require sample preparation. We use the Advanced Multimodality Image Guided Operating Suite at Brigham and Women’s Hospital to demonstrate that desorption electrospray ionization MS could be used to detect residual tumor that would have been left behind in the patient. The approach paves the way for the clinical testing of MS-based intraoperative monitoring of tumor metabolites, an advance that could revolutionize the care of surgical oncology patients. For many intraoperative decisions surgeons depend on frozen section pathology, a technique developed over 150 y ago. Technical innovations that permit rapid molecular characterization of tissue samples at the time of surgery are needed. Here, using desorption electrospray ionization (DESI) MS, we rapidly detect the tumor metabolite 2-hydroxyglutarate (2-HG) from tissue sections of surgically resected gliomas, under ambient conditions and without complex or time-consuming preparation. With DESI MS, we identify isocitrate dehydrogenase 1-mutant tumors with both high sensitivity and specificity within minutes, immediately providing critical diagnostic, prognostic, and predictive information. Imaging tissue sections with DESI MS shows that the 2-HG signal overlaps with areas of tumor and that 2-HG levels correlate with tumor content, thereby indicating tumor margins. Mapping the 2-HG signal onto 3D MRI reconstructions of tumors allows the integration of molecular and radiologic information for enhanced clinical decision making. We also validate the methodology and its deployment in the operating room: We have installed a mass spectrometer in our Advanced Multimodality Image Guided Operating (AMIGO) suite and demonstrate the molecular analysis of surgical tissue during brain surgery. This work indicates that metabolite-imaging MS could transform many aspects of surgical care.

Multicolored stain-free histopathology with coherent Raman imaging

Conventional histopathology with hematoxylin & eosin (H&E) has been the gold standard for histopathological diagnosis of a wide range of diseases. However, it is not performed in vivo and requires thin tissue sections obtained after tissue biopsy, which carries risk, particularly in the central nervous system. Here we describe the development of an alternative, multicolored way to visualize tissue in real-time through the use of coherent Raman imaging (CRI), without the use of dyes. CRI relies on intrinsic chemical contrast based on vibrational properties of molecules and intrinsic optical sectioning by nonlinear excitation. We demonstrate that multicolor images originating from CH2 and CH3 vibrations of lipids and protein, as well as two-photon absorption of hemoglobin, can be obtained with subcellular resolution from fresh tissue. These stain-free histopathological images show resolutions similar to those obtained by conventional techniques, but do not require tissue fixation, sectioning or staining of the tissue analyzed.

Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy

Quantitative SRS microscopy can detect human brain tumor infiltration with high sensitivity and specificity, even in tissues appearing grossly normal. Image-based classifier calls out cancer cells Ji and colleagues used a microscopy technique called stimulated Raman scattering, or SRS, to image cancer cells in human brain tissue. SRS produces different signals for proteins and lipids, which can then be assigned a color (blue and green, respectively), allowing the authors to differentiate brain cortex from tumor from white matter. Biopsies from adult and pediatric patients with glioblastoma revealed not only distinctive features with SRS microscopy but also the presence of infiltrating cells in tissues that appeared otherwise normal with traditional staining. Such infiltrating cells are important to catch early because leaving them behind after surgery nearly always leads to cancer recurrence. To make this SRS microscopy approach amenable to routine use in neuropathology, the authors also created an objective classifier that integrated different image characteristics, such as the protein/lipid ratio, axonal density, and degree of cellularity, into one output, on a scale of 0 to 1, that would alert the pathologist to tumor infiltration. The classifier was built using more than 1400 images from patients with glioblastoma and epilepsy, and could distinguish between tumor-infiltrated and nontumor regions with >99% accuracy, regardless of tumor grade or histologic subtype. This label-free imaging technology could therefore be used to complement existing neurosurgical workflows, allowing for rapid and objective characterization of brain tissues and, in turn, clinical decision-making. Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a nondestructive, label-free optical method, to reveal glioma infiltration in animal models. We show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ = 0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density, and protein/lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density, and protein/lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery.

Methylene Blue-Conjugated Hydrogel Nanoparticles and Tumor-Cell Targeted Photodynamic Therapy

Methylene blue-conjugated polyacrylamide nanoparticles are prepared through a microemulsion polymerization, after conjugation of the dye with a monomer. The nanoparticles have a 50-60 nm diameter in solution. This conjugation method enables a large increase in loading of methylene blue per nanoparticle and also minimizes dye leaching out of the nanoparticle. Furthermore, the dye content can be controlled by variation of the dye amount, enabling a more refined control of the singlet oxygen production ability. The nanoparticles are coated with F3 peptides, which give specific targeting to selected tumor cells, 9L, MDA-MB-435, and F98, in vitro. In addition, MTT assays reveal that the nanoparticles have no dark toxicity but excellent PDT efficacy increasing with the nanoparticle dose and irradiation time.

Small Solutions for Big Problems: The Application of Nanoparticles to Brain Tumor Diagnosis and Therapy

Nanotechnology has been projected to have a significant impact on the future treatment of brain tumors. Specifically, nanoparticles have the potential to revolutionize brain tumor imaging as well as surgical and adjuvant treatments. The translation of current research in nanotechnology into clinical practice will rely on solving challenges relating to the pharmacology of nanoparticles.

Neuronavigation in the surgical management of brain tumors: current and future trends

Neuronavigation has become an ubiquitous tool in the surgical management of brain tumors. This review describes the use and limitations of current neuronavigational systems for brain tumor biopsy and resection. Methods for integrating intraoperative imaging into neuronavigational datasets developed to address the diminishing accuracy of positional information that occurs over the course of brain tumor resection are discussed. In addition, the process of integration of functional MRI and tractography into navigational models is reviewed. Finally, emerging concepts and future challenges relating to the development and implementation of experimental imaging technologies in the navigational environment are explored.

In vitro characterization of a targeted, dye-loaded nanodevice for intraoperative tumor delineation.

OBJECTIVETo synthesize and complete in vitro characterization of a novel, tumor-targeted nanodevice for visible intraoperative delineation of brain tumors. METHODSThe ability of dye-loaded polyacrylamide nanoparticles (NP) containing methylene blue, Coomassie blue, or indocyanine green to cause color change in the 9L glioma cell lines was evaluated. Cells were incubated with dye-loaded NPs, photographed, and analyzed colorimetrically. Confocal microscopy was used to determine subcellular localization of NPs in treated cells. RESULTSIncubation of glioma cell lines with dye-loaded NPs resulted in clearly visible, quantifiable cell tagging in a dose- and time-dependent manner. Dye-loaded NPs were observed to bind to the surface and become internalized by glioma cells. Coating the NP surface with F3, a peptide that binds to the tumor cell surface receptor nucleolin, significantly increased NP affinity for glioma cells. F3 targeting also significantly increased the rate of cell tagging by dye-loaded NPs. Finally, F3-targeted NPs demonstrated specificity for targeting various cancer cell lines based on their surface expression of cell surface nucleolin. CONCLUSIONF3-targeted dye-loaded NPs efficiently cause definitive color change in glioma cells. This report represents the first use of targeted NPs to cause a visible color change in tumor cell lines. Similar nanodevices may be used in the future to enable visible intraoperative tumor delineation during tumor resection.

Mechanisms of Glioma Formation: Iterative Perivascular Glioma Growth and Invasion Leads to Tumor Progression, VEGF-Independent Vascularization, and Resistance to Antiangiogenic Therapy

As glioma cells infiltrate the brain they become associated with various microanatomic brain structures such as blood vessels, white matter tracts, and brain parenchyma. How these distinct invasion patterns coordinate tumor growth and influence clinical outcomes remain poorly understood. We have investigated how perivascular growth affects glioma growth patterning and response to antiangiogenic therapy within the highly vascularized brain. Orthotopically implanted rodent and human glioma cells are shown to commonly invade and proliferate within brain perivascular space. This form of brain tumor growth and invasion is also shown to characterize de novo generated endogenous mouse brain tumors, biopsies of primary human glioblastoma (GBM), and peripheral cancer metastasis to the human brain. Perivascularly invading brain tumors become vascularized by normal brain microvessels as individual glioma cells use perivascular space as a conduit for tumor invasion. Agent-based computational modeling recapitulated biological perivascular glioma growth without the need for neoangiogenesis. We tested the requirement for neoangiogenesis in perivascular glioma by treating animals with angiogenesis inhibitors bevacizumab and DC101. These inhibitors induced the expected vessel normalization, yet failed to reduce tumor growth or improve survival of mice bearing orthotopic or endogenous gliomas while exacerbating brain tumor invasion. Our results provide compelling experimental evidence in support of the recently described failure of clinically used antiangiogenics to extend the overall survival of human GBM patients.