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

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.

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.

Macrophage-conditioned medium inhibits the activation of cyclin-dependent kinase 2 by adipogenic inducers in 3T3-L1 preadipocytes.

Macrophage infiltration into adipose tissue, associated with obesity, is thought to contribute to abnormal adipose tissue remodeling, low-grade inflammation, and insulin resistance. Medium conditioned by macrophages (MacCM) inhibits 3T3-L1 and human adipocyte differentiation, as well as early adipogenic cell cycle events including MCE and retinoblastoma protein (Rb) phosphorylation. Our objective was to determine if the inhibition of Rb phosphorylation was linked to changes in cell cycle-related proteins. We treated 3T3-L1 preadipocytes with adipogenic inducers for 24 h in control medium versus J774A.1-MacCM. The differentiation-induced mRNA and protein expression of cyclin A, an activator of cyclin-dependent kinase (cdk) 2 which phosphorylates Rb, was inhibited by 82% and 73%, respectively, by J774A.1-MacCM; adipogenic expression of Myc, a transcriptional regulator of cyclin A, was also suppressed significantly. Consistent with the reduction in cyclin A levels, the activation of cdk2 by adipogenic inducers was inhibited by 75% by J774A.1-MacCM. J774A.1-MacCM also lowered levels of cyclins D1 and D2. Inhibition studies demonstrated that platelet-derived growth factor, an anti-adipogenic factor found in J774A.1-MacCM, was not responsible for the inhibitory effect on differentiation. The anti-adipogenic effect of J774A.1-MacCM was resistant to proteinase K and heat treatment, and was present in a <3 kDa fraction. Our data indicate that J774A.1-MacCM interferes with the upregulation of cyclin A levels and cdk2 activity that are required for Rb phosphorylation and MCE in 3T3-L1 adipogenesis.

Mass spectrometry imaging as a tool for surgical decision-making.

Despite significant advances in image-guided therapy, surgeons are still too often left with uncertainty when deciding to remove tissue. This binary decision between removing and leaving tissue during surgery implies that the surgeon should be able to distinguish tumor from healthy tissue. In neurosurgery, current image-guidance approaches such as magnetic resonance imaging (MRI) combined with neuronavigation offer a map as to where the tumor should be, but the only definitive method to characterize the tissue at stake is histopathology. Although extremely valuable information is derived from this gold standard approach, it is limited to very few samples during surgery and is not practically used for the delineation of tumor margins. The development and implementation of faster, comprehensive, and complementary approaches for tissue characterization are required to support surgical decision-making--an incremental and iterative process with tumor removed in multiple and often minute biopsies. The development of atmospheric pressure ionization sources makes it possible to analyze tissue specimens with little to no sample preparation. Here, we highlight the value of desorption electrospray ionization as one of many available approaches for the analysis of surgical tissue. Twelve surgical samples resected from a patient during surgery were analyzed and diagnosed as glioblastoma tumor or necrotic tissue by standard histopathology, and mass spectrometry results were further correlated to histopathology for critical validation of the approach. The use of a robust statistical approach reiterated results from the qualitative detection of potential biomarkers of these tissue types. The correlation of the mass spectrometry and histopathology results to MRI brings significant insight into tumor presentation that could not only serve to guide tumor resection, but that is also worthy of more detailed studies on our understanding of tumor presentation on MRI.

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.