Multimodal platform for assessing drug distribution and response in clinical trials.

BACKGROUND: Response to targeted therapy varies between patients for largely unknown reasons. Here, we developed and applied an integrative platform using mass spectrometry imaging (MSI), phosphoproteomics, and multiplexed tissue imaging for mapping drug distribution, target engagement, and adaptive response to gain insights into heterogeneous response to therapy. METHODS: Patient-derived xenograft (PDX) lines of glioblastoma were treated with adavosertib, a Wee1 inhibitor, and tissue drug distribution was measured with MALDI-MSI. Phosphoproteomics was measured in the same tumors to identify biomarkers of drug target engagement and cellular adaptive response. Multiplexed tissue imaging was performed on sister sections to evaluate spatial co-localization of drug and cellular response. The integrated platform was then applied on clinical specimens from glioblastoma patients enrolled in the phase 1 clinical trial. RESULTS: PDX tumors exposed to different doses of adavosertib revealed intra- and inter-tumoral heterogeneity of drug distribution and integration of the heterogeneous drug distribution with phosphoproteomics and multiplexed tissue imaging revealed new markers of molecular response to adavosertib. Analysis of paired clinical specimens from patients enrolled in the phase 1 clinical trial informed the translational potential of the identified biomarkers in studying patient's response to adavosertib. CONCLUSIONS: The multimodal platform identified a signature of drug efficacy and patient-specific adaptive responses applicable to preclinical and clinical drug development. The information generated by the approach may inform mechanisms of success and failure in future early phase clinical trials, providing information for optimizing clinical trial design and guiding future application into clinical practice.

Spatial Distribution of Transcytosis Relevant Phospholipids in Response to Omega-3 Dietary Deprivation.

The cell membrane of brain endothelial cells is enriched in omega-3 phospholipid species. Numerous omega-3 phospholipid species were recently proposed to be important for maintaining the low rate of transcytosis and, thus, could be important for regulating one of the mechanisms of the blood brain barrier (BBB). However, the spatial distribution of these phospholipid species within the brain was previously unknown. Here, we combined advanced mass spectrometry imaging techniques to generate a map of these phospholipids in the brain at near single cell resolution. Furthermore, we explored the effects of omega-3 dietary deprivation on both docosahexaenoic acid (DHA)-containing phospholipids and the global brain phospholipid profile. We demonstrate the unique spatial distribution of individual DHA-containing phospholipids, which may be important for the regiospecific properties of the BBB. Finally, 24 diet discriminative phospholipids were identified and showed an increase in saturated phospholipid species and ceramide containing phospholipid species under omega-3 dietary deficiency.

Rapid MALDI mass spectrometry imaging for surgical pathology.

Matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is an emerging analytical technique, which generates spatially resolved proteomic and metabolomic images from tissue specimens. Conventional MALDI MSI processing and data acquisition can take over 30 min, limiting its clinical utility for intraoperative diagnostics. We present a rapid MALDI MSI method, completed under 5 min, including sample preparation and analysis, providing a workflow compatible with the clinical frozen section procedure.

Metal Oxide Laser Ionization Mass Spectrometry Imaging (MOLI MSI) Using Cerium(IV) Oxide.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a powerful technique for spatially resolved metabolomics. A variation on MALDI, termed metal oxide laser ionization (MOLI), capitalizes on the unique property of cerium(IV) oxide (CeO2) to induce laser-catalyzed fatty acyl cleavage from lipids and has been utilized for bacterial identification. In this study, we present the development and utilization of CeO2 as an MSI catalyst. The method was developed using a MALDI TOF instrument in negative ion mode, equipped with a high frequency laser. Instrument parameters for MOLI MS fatty acid catalysis with CeO2 were optimized with phospholipid standards and fatty acid catalysis was confirmed using lipid extracts from reference bacterial strains, and sample preparation was optimized using mouse brain tissue. MOLI MSI was applied to the imaging of normal mouse brain revealing differentiable fatty acyl pools in myelinated and nonmyelinated regions. Similarly, MOLI MSI showed distinct fatty acyl composition in tumor regions of a patient derived xenograft mouse model of glioblastoma. To assess the potential of MOLI MSI to detect pathogens directly from tissue, a pseudoinfection model was prepared by spotting Escherichia coli lipid extracts on mouse brain tissue sections and imaged by MOLI MSI. The spotted regions were molecularly resolved from the supporting mouse brain tissue by the diagnostic odd-chained fatty acids and reflected control bacterial MOLI MS signatures. We describe MOLI MSI for the first time and highlight its potential for spatially resolved fatty acyl analysis, characterization of fatty acyl composition in tumors, and its potential for pathogen detection directly from tissue.

Rapid discrimination of pediatric brain tumors by mass spectrometry imaging.

PURPOSE: Medulloblastoma, the most common primary pediatric malignant brain tumor, originates in the posterior fossa of the brain. Pineoblastoma, which originates within the pineal gland, is a rarer malignancy that also presents in the pediatric population. Medulloblastoma and pineoblastoma exhibit overlapping clinical features and have similar histopathological characteristics. Histopathological similarities confound rapid diagnoses of these two tumor types. We have conducted a pilot feasibility study analyzing the molecular profile of archived frozen human tumor specimens using mass spectrometry imaging (MSI) to identify potential biomarkers capable of classifying and distinguishing between medulloblastoma and pineoblastoma. METHODS: We performed matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry imaging on eight medulloblastoma biopsy specimens and three pineoblastoma biopsy specimens. Multivariate statistical analyses were performed on the MSI dataset to generate classifiers that distinguish the two tumor types. Lastly, the molecules that were discriminative of tumor type were queried against the Lipid Maps database and identified. RESULTS: In this pilot study we show that medulloblastoma and pineoblastoma can be discriminated using molecular profiles determined by MSI. The highest-ranking discriminating classifiers of medulloblastoma and pineoblastoma were glycerophosphoglycerols and sphingolipids, respectively. CONCLUSION: We demonstrate proof-of-concept that medulloblastoma and pineoblastoma can be rapidly distinguished by using MSI lipid profiles. We identified biomarker candidates capable of distinguishing these two histopathologically similar tumor types. This work expands the current molecular knowledge of medulloblastoma and pineoblastoma by characterizing their lipidomic profiles, which may be useful for developing novel diagnostic, prognostic and therapeutic strategies.

In Vitro Liquid Extraction Surface Analysis Mass Spectrometry (ivLESA-MS) for Direct Metabolic Analysis of Adherent Cells in Culture.

Conventional metabolomic methods include extensive sample preparation steps and long analytical run times, increasing the likelihood of processing artifacts and limiting high throughput applications. We present here in vitro liquid extraction surface analysis mass spectrometry (ivLESA-MS), a variation on LESA-MS, performed directly on adherent cells grown in 96-well cell culture plates. To accomplish this, culture medium was aspirated immediately prior to analysis, and metabolites were extracted using LESA from the cell monolayer surface, followed by nano-electrospray ionization and MS analysis in negative ion mode. We applied this platform to characterize and compare lipidomic profiles of multiple breast cancer cell lines growing in culture (MCF-7, ZR-75-1, MDA-MB-453, and MDA-MB-231) and revealed distinct and reproducible lipidomic signatures between the cell lines. Additionally, we demonstrated time-dependent processing artifacts, underscoring the importance of immediate analysis. ivLESA-MS represents a rapid in vitro metabolomic method, which precludes the need for quenching, cell harvesting, sample preparation, and chromatography, significantly shortening preparation and analysis time while minimizing processing artifacts. This method could be further adapted to test drugs in vitro in a high throughput manner.

In Vivo Neural Tissue Engineering: Cylindrical Biocompatible Hydrogels That Create New Neural Tracts in the Adult Mammalian Brain.

Individuals with neurodegenerative disorders or brain injury have few treatment options and it has been proposed that endogenous adult neural stem cells can be harnessed to repopulate dysfunctional nonneurogenic regions of the brain. We have accomplished this through the development of rationally designed hydrogel implants that recruit endogenous cells from the adult subventricular zone to create new relatively long tracts of neuroblasts. These implants are biocompatible and biodegradable cylindrical hydrogels consisting of fibrin and immobilized neurotrophic factors. When implanted into rat brain such that the cylinder intersected the migratory path of endogenous neural progenitors (the rostral migratory stream) and led into the nonneurogenic striatum, we observed a robust neurogenic response in the form of migrating neuroblasts with long (>100 µm) complex neurites. The location of these new neural cells in the striatum was directly coincident with the original track of the fibrin implant, which itself had completely degraded, and covered a significant area and distance (>2.5 mm). We also observed a significant number of neuroblasts in the striatal region between the implant track and the lateral ventricle. When these fibrin cylinders were implanted into hemiparkinson rats, correction of parkinsonian behavior was observed. There were no obvious behavioral, inflammatory or tumorigenic sequelae as a consequence of the implants. In conclusion, we have successfully engineered neural tissue in vivo, using neurogenic biomaterials cast into a unique cylindrical architecture. These results represent a novel approach to efficiently induce neurogenesis in a controlled and targeted manner, which may lead toward a new therapeutic modality for neurological disorders.

Effect of electron donating groups on polyphenol-based antioxidant dendrimers.

Numerous studies have reported the beneficial effects of antioxidants in human diseases. Among their biological effects, a majority of antioxidants scavenge reactive radicals in the body, thereby reducing oxidative stress that is associated with the pathogenesis of many diseases. Antioxidant dendrimers are a new class of potent antioxidant compounds reported recently. In this study, six polyphenol-based antioxidant dendrimers with or without electron donating groups (methoxy group) were synthesized in order to elucidate the influence of electron donating groups (EDG) on their antioxidant activities. Syringaldehyde (2 ortho methoxy groups), vanillin (1 ortho methoxy group), and 4-hydroxybenzaldehyde (0 methoxy group) were derivatized with propargylamine to form building blocks for the dendrimers. All the six dendrimers contain polyether cores, which were synthesized by attaching pentaerythritol and methyl α-d-glucopyranoside to in-house prepared spacer units. To prepare generation 1 antioxidant dendrimers, microwave energy and granulated metallic copper catalyst were used to link the cores and building blocks together via alkyne-azide 1,3-cycloaddition click chemistry. These reaction conditions resulted in high yields of the target dendrimers that were free from copper contamination. Based on DPPH antioxidant assay, antioxidant dendrimers decorated with syringaldehyde and vanillin exhibited over 70- and 170-fold increase in antioxidant activity compared to syringaldehyde and vanillin, respectively. The antioxidant activity of dendrimers increased with increasing number of EDG groups. Similar results were obtained when the dendrimers were used to protect DNA and human LDL against organic carbon and nitrogen-based free radicals. In addition, the antioxidant dendrimers did not show any pro-oxidant activity on DNA in the presence of physiological amounts of copper. Although the dendrimers showed potent antioxidant activities against carbon and nitrogen free radicals, EPR and DNA protection studies revealed lack of effectiveness of these dendrimers against hydroxyl radicals. The dendrimers were not cytotoxic to CHO-K1 cells.