Nathalie Y. R. Agar

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.

Classifying Human Brain Tumors by Lipid Imaging with Mass Spectrometry

Brain tissue biopsies are required to histologically diagnose brain tumors, but current approaches are limited by tissue characterization at the time of surgery. Emerging technologies such as mass spectrometry imaging can enable a rapid direct analysis of cancerous tissue based on molecular composition. Here, we illustrate how gliomas can be rapidly classified by desorption electrospray ionization-mass spectrometry (DESI-MS) imaging, multivariate statistical analysis, and machine learning. DESI-MS imaging was carried out on 36 human glioma samples, including oligodendroglioma, astrocytoma, and oligoastrocytoma, all of different histologic grades and varied tumor cell concentration. Gray and white matter from glial tumors were readily discriminated and detailed diagnostic information could be provided. Classifiers for subtype, grade, and concentration features generated with lipidomic data showed high recognition capability with more than 97% cross-validation. Specimen classification in an independent validation set agreed with expert histopathology diagnosis for 79% of tested features. Together, our findings offer proof of concept that intraoperative examination and classification of brain tissue by mass spectrometry can provide surgeons, pathologists, and oncologists with critical and previously unavailable information to rapidly guide surgical resections that can improve management of patients with malignant brain tumors.

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.

Protein aggregation and protein instability govern familial amyotrophic lateral sclerosis patient survival.

The nature of the “toxic gain of function” that results from amyotrophic lateral sclerosis (ALS)-, Parkinson-, and Alzheimer-related mutations is a matter of debate. As a result no adequate model of any neurodegenerative disease etiology exists. We demonstrate that two synergistic properties, namely, increased protein aggregation propensity (increased likelihood that an unfolded protein will aggregate) and decreased protein stability (increased likelihood that a protein will unfold), are central to ALS etiology. Taken together these properties account for 69% of the variability in mutant Cu/Zn-superoxide-dismutase-linked familial ALS patient survival times. Aggregation is a concentration-dependent process, and spinal cord motor neurons have higher concentrations of Cu/Zn-superoxide dismutase than the surrounding cells. Protein aggregation therefore is expected to contribute to the selective vulnerability of motor neurons in familial ALS.

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.

Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis

Significance This study is the first demonstration, to our knowledge, of the application of desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for discrimination of breast cancer and delineation of tumor margins. Using DESI-MSI, it is possible to discriminate between cancerous and adjacent normal tissue on the basis of the detection and specific spatial distributions of different lipid species. This study proves the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. It will allow the surgeon to access to this information in real time so as to make accurate intraoperative decisions quickly. It will result in improved cosmesis and decrease the need for multiple operations for margin reexcision. Distinguishing tumor from normal glandular breast tissue is an important step in breast-conserving surgery. Because this distinction can be challenging in the operative setting, up to 40% of patients require an additional operation when traditional approaches are used. Here, we present a proof-of-concept study to determine the feasibility of using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for identifying and differentiating tumor from normal breast tissue. We show that tumor margins can be identified using the spatial distributions and varying intensities of different lipids. Several fatty acids, including oleic acid, were more abundant in the cancerous tissue than in normal tissues. The cancer margins delineated by the molecular images from DESI-MSI were consistent with those margins obtained from histological staining. Our findings prove the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. The results suggest that an MS-based method could be developed for the rapid intraoperative detection of residual cancer tissue during breast-conserving surgery.

Desorption Electrospray Ionization then MALDI Mass Spectrometry Imaging of Lipid and Protein Distributions in Single Tissue Sections

Imaging mass spectrometry (MS) is a powerful technique for mapping the spatial distributions of a wide range of chemical compounds simultaneously from a tissue section. Co-localization of the distribution of individual molecular species, including particular lipids and proteins, and correlation with the morphological features of a single tissue section are highly desirable for comprehensive tissue analysis and disease diagnosis. We now report on the use, in turn, of desorption electrospray ionization (DESI), matrix assisted laser desorption ionization (MALDI), and then optical microscopy to image lipid and protein distributions in a single tissue section. This is possible through the use of histologically compatible DESI solvent systems, which allow for sequential analyses of the same section by DESI then MALDI. Hematoxylin and eosin (H&E) staining was performed on the same section after removal of the MALDI matrix. This workflow allowed chemical information to be unambiguously matched to histological features in mouse brain tissue sections. The lipid sulfatide (24:1), detected at m/z 888.8 by DESI imaging, was colocalized with the protein MBP isoform 8, detected at m/z 14117 by MALDI imaging, in regions corresponding to the corpus callosum substructure of the mouse brain, as confirmed in the H&E images. Correlation of lipid and protein distributions with histopathological features was also achieved for human brain cancer samples. Higher tumor cell density was observed in regions demonstrating higher relative abundances of oleic acid, detected by DESI imaging at m/z 281.4, and the protein calcyclin, detected by MALDI at m/z 10085, for a human glioma sample. Since correlation between molecular signatures and disease state can be achieved, we expect that this methodology will significantly enhance the value of MS imaging in molecular pathology for diagnosis.

The Imprinted Gene PEG3 Inhibits Wnt Signaling and Regulates Glioma Growth

The imprinted gene PEG3 confers parenting and sexual behaviors, alters growth and development, and regulates apoptosis. However, a molecular mechanism that can account for the diverse functions of Peg3/Pw1 is not known. To elucidate Peg3-regulated pathways, we performed a functional screen in zebrafish. Enforced overexpression of PEG3 mRNA during zebrafish embryogenesis decreased β-catenin protein expression and inhibited Wnt-dependent tail development. Peg3/Pw1 also inhibited Wnt signaling in human cells by binding to β-catenin and promoting its degradation via a p53/Siah1-dependent, GSK3β-independent proteasomal pathway. The inhibition of the Wnt pathway by Peg3/Pw1 suggested a role in tumor suppression. Hypermethylation of the PEG3 promoter in primary human gliomas led to a loss of imprinting and decreased PEG3 mRNA expression that correlated with tumor grade. The decrease in Peg3/Pw1 protein expression increased β-catenin, promoted proliferation, and inhibited p53-dependent apoptosis in human CD133+ glioma stem cells. Thus, mammalian imprinting utilizes Peg3/Pw1 to co-opt the Wnt pathway, thereby regulating development and glioma growth.

Molecular imaging of drug transit through the blood-brain barrier with MALDI mass spectrometry imaging

Drug transit through the blood-brain barrier (BBB) is essential for therapeutic responses in malignant glioma. Conventional methods for assessment of BBB penetrance require synthesis of isotopically labeled drug derivatives. Here, we report a new methodology using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) to visualize drug penetration in brain tissue without molecular labeling. In studies summarized here, we first validate heme as a simple and robust MALDI MSI marker for the lumen of blood vessels in the brain. We go on to provide three examples of how MALDI MSI can provide chemical and biological insights into BBB penetrance and metabolism of small molecule signal transduction inhibitors in the brain – insights that would be difficult or impossible to extract by use of radiolabeled compounds.

Inhibition of medulloblastoma cell invasion by Slit

Invasion of brain tumor cells has made primary malignant brain neoplasms among the most recalcitrant to therapeutic strategies. We tested whether the secreted protein Slit2, which guides the projection of axons and developing neurons, could modulate brain tumor cell invasion. Slit2 inhibited the invasion of medulloblastoma cells in a variety of in vitro models. The effect of Slit2 was inhibited by the Robo ectodomain. Time-lapse videomicroscopy indicated that Slit2 reduced medulloblastoma invasion rate without affecting cell direction or proliferation. Both medulloblastoma and glioma tumors express Robo1 and Slit2, but only medulloblastoma invasion is inhibited by recombinant Slit2 protein. Downregulation of activated Cdc42 may contribute to this differential response. Our findings reinforce the concept that neurodevelopmental cues such as Slit2 may provide insights into brain tumor invasion.