Spatial intra-tumor heterogeneity is associated with survival of lung adenocarcinoma patients.

Intra-tumor heterogeneity (ITH) of human tumors is important for tumor progression, treatment response, and drug resistance. However, the spatial distribution of ITH remains incompletely understood. Here, we present spatial analysis of ITH in lung adenocarcinomas from 147 patients using multi-region mass spectrometry of >5,000 regions, single-cell copy number sequencing of ~2,000 single cells, and cyclic immunofluorescence of >10 million cells. We identified two distinct spatial patterns among tumors, termed clustered and random geographic diversification (GD). These patterns were observed in the same samples using both proteomic and genomic data. The random proteomic GD pattern, which is characterized by decreased cell adhesion and lower levels of tumor-interacting endothelial cells, was significantly associated with increased risk of recurrence or death in two independent patient cohorts. Our study presents comprehensive spatial mapping of ITH in lung adenocarcinoma and provides insights into the mechanisms and clinical consequences of GD.

Biomarker Mapping on Skin Tape Strips Using MALDI Mass Spectrometry Imaging.

Keratinocyte organization and biochemistry are important in forming the skin's protective barrier. Intrinsic and extrinsic factors can affect skin barrier function at the cellular and molecular levels. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging, a technique which combines both molecular aspects and histological details, has proven to be a valuable method in various disciplines including pharmacology, dermatology and cosmetology. It typically requires ex vivo samples, prepared following frozen tissue sectioning. This paper demonstrates the feasibility of performing MALDI analysis on tape strips collected non-invasively on skin. The aim is to obtain molecular imaging of corneocytes on tapes towards novel biological insights. Tapes were collected from two skin sites (volar forearm and cheek) of human volunteers. Ten molecules relating to skin barrier function were detected with a single mode of acquisition at high spatial resolution with a 7 T MALDI-Fourier transform ion cyclotron resonance (FTICR) instrument. The method sensitivity was adequate to create molecular maps which could be overlaid on transmission microscopy images of the same area of the tape. Analysis of the molecular distributions from tapes at the two skin sites was consistent with the known skin properties of the two sites, confirming the validity of the observations. Hierarchical clustering analysis was used to differentiate corneocyte populations based on their molecular profiles. Furthermore, morphological analysis provided a new way of considering statistical populations of corneocytes on the same tape, rather than measuring a single averaged value, providing additional useful information relating to their structure-function relationship.

Application of a self-assembling peptide matrix prevents esophageal stricture after circumferential endoscopic submucosal dissection in a pig model.

INTRODUCTION: Circumferential endoscopic submucosal dissection (ESD) allows to treat large esophageal superficial neoplasms, however with a high occurrence of severe esophageal strictures. In a previous work, we demonstrated that the application of a prototype of self-assembling peptide (SAP) matrix on esophageal wounds after a circumferential-ESD delayed the onset of esophageal stricture in a porcine model. The aim of this work was to consolidate these results using the commercialized version of this SAP matrix currently used as a hemostatic agent. ANIMALS AND METHODS: Eleven pigs underwent a 5 cm-long circumferential esophageal ESD under general anesthesia. Five pigs were used as a control group and six were treated with the SAP. In the experimental group, 3.5 mL of the SAP matrix were immediately applied on the ESD wound. Stricture rates and esophageal diameter were assessed at day 14 by endoscopy and esophagram, followed by necropsy and histological measurements of inflammation and fibrosis in the esophageal wall. RESULTS: At day 14, two animals in the treated group had an esophageal stricture without any symptom, while all animals in the control group had regurgitations and an esophageal stricture (33 vs. 100%, p = 0.045). In the treated group, the mean esophageal diameter at day 14 was 9.5 ± 1 mm vs. 4 ± 0.6 mm in the control group (p = 0.004). Histologically, the neoepithelium was longer in the SAP treated group vs. the control (3075 µm vs. 1155µm, p = 0.014). On immunohistochemistry, the expression of alpha smooth muscle actin was lower in the treated vs. control group. CONCLUSION: Apposition of a self-assembling peptide matrix immediately after a circumferential esophageal ESD reduced by 67% the occurrence of a stricture at day 14, by promoting reepithelialization of the resected area.

Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging of Key Proteins in Corneal Samples from Lattice Dystrophy Patients with TGFBI-H626R and TGFBI-R124C Mutations.

SCOPE: The purpose of this study is to identify and visualize the spatial distribution of proteins present in amyloid corneal deposits of TGFBI-CD patients using Mass Spectrometry Imaging (MSI) and compare it with healthy control cornea. Corneal Dystrophies (CD) constitute a group of genetically inherited protein aggregation disorders that affects different layers of the cornea. With accumulated protein deposition, the cornea becomes opaque with decreased visual acuity. CD affecting the stroma and Bowman's membrane, is associated with mutations in transforming growth factor ß-induced (TGFBI) gene. METHODS: MALDI-Mass Spectrometry Imaging (MSI) is performed on 2 patient corneas and is compared with 1 healthy control cornea using a 7T-MALDI-FTICR. Molecular images obtained are overlaid with congo-red stained sections to visualize the proteins associated with the corneal amyloid aggregates. RESULTS: MALDI-MSI provides a relative abundance and two dimensional spatial protein signature of key proteins (TGFBIp, Apolipoprotein A-I, Apolipoprotein A-IV, Apolipoprotein E, Kaliocin-1, Pyruvate Kinase and Ras related protein Rab-10) in the patient deposits compared to the control. This is the first report of the anatomical localization of key proteins on corneal tissue section from CD patients. This may provide insight in understanding the mechanism of amyloid fibril formation in TGFBI-corneal dystrophy.

Pharmacokinetics and Skin Tolerability of Intracutaneous Zolmitriptan Delivery in Swine Using Adhesive Dermally Applied Microarray.

Adhesive Dermally Applied Microarray (ADAM) is a new drug-delivery system that uses microprojections (340-µm long) for intracutaneous drug self-administration. We formulated zolmitriptan, a well-accepted and commonly used migraine medication, for administration using ADAM. In vivo studies were conducted in female prepubescent Yorkshire pigs using ADAM 1.9-mg zolmitriptan applied to the inner thigh and left in place for 1 h. Pharmacokinetic studies showed that the ADAM 1.9-mg zolmitriptan was delivered with high efficiency (85%) and high absolute bioavailability (77%). Furthermore, in vivo evaluation showed a rapid systemic absorption with a median Tmax of 15 min. Skin biopsies of the treatment sites showed a mean depth of microprojection penetration of 105.4 ± 3.6 µm. Mass spectrometry imaging showed that the zolmitriptan after 1 h of patch wear time was predominantly localized to the dermis. ADAM zolmitriptan was well tolerated with a transient mild-to-moderate erythema response. The findings in these studies, particularly the rapid zolmitriptan absorption profile after intracutaneous administration, provided validation to advance ADAM zolmitriptan development.

Altered metabolic landscape in IDH-mutant gliomas affects phospholipid, energy, and oxidative stress pathways.

Heterozygous mutations in NADP-dependent isocitrate dehydrogenases (IDH) define the large majority of diffuse gliomas and are associated with hypermethylation of DNA and chromatin. The metabolic dysregulations imposed by these mutations, whether dependent or not on the oncometabolite D-2-hydroxyglutarate (D2HG), are less well understood. Here, we applied mass spectrometry imaging on intracranial patient-derived xenografts of IDH-mutant versus IDH wild-type glioma to profile the distribution of metabolites at high anatomical resolution in situ This approach was complemented by in vivo tracing of labeled nutrients followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Selected metabolites were verified on clinical specimen. Our data identify remarkable differences in the phospholipid composition of gliomas harboring the IDH1 mutation. Moreover, we show that these tumors are characterized by reduced glucose turnover and a lower energy potential, correlating with their reduced aggressivity. Despite these differences, our data also show that D2HG overproduction does not result in a global aberration of the central carbon metabolism, indicating strong adaptive mechanisms at hand. Intriguingly, D2HG shows no quantitatively important glucose-derived label in IDH-mutant tumors, which suggests that the synthesis of this oncometabolite may rely on alternative carbon sources. Despite a reduction in NADPH, glutathione levels are maintained. We found that genes coding for key enzymes in de novo glutathione synthesis are highly expressed in IDH-mutant gliomas and the expression of cystathionine-ß-synthase (CBS) correlates with patient survival in the oligodendroglial subtype. This study provides a detailed and clinically relevant insight into the in vivo metabolism of IDH1-mutant gliomas and points to novel metabolic vulnerabilities in these tumors.

Chloroquine and chloroquinoline derivatives as models for the design of modulators of amyloid Peptide precursor metabolism.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD). Preventing deregulated APP processing by inhibiting amyloidogenic processing of carboxy-terminal fragments (APP-CTFs), and reducing the toxic effect of amyloid beta (Aß) peptides remain an effective therapeutic strategy. We report the design of piperazine-containing compounds derived from chloroquine structure and evaluation of their effects on APP metabolism and ability to modulate the processing of APP-CTF and the production of Aß peptide. Compounds which retained alkaline properties and high affinity for acidic cell compartments were the most effective. The present study demonstrates that (1) the amino side chain of chloroquine can be efficiently substituted by a bis(alkylamino)piperazine chain, (2) the quinoline nucleus can be replaced by a benzyl or a benzimidazole moiety, and (3) pharmacomodulation of the chemical structure allows the redirection of APP metabolism toward a decrease of Aß peptide release, and increased stability of APP-CTFs and amyloid intracellular fragment. Moreover, the benzimidazole compound 29 increases APP-CTFs in vivo and shows promising activity by the oral route. Together, this family of compounds retains a lysosomotropic activity which inhibits lysosome-related Aß production, and is likely to be beneficial for therapeutic applications in AD.

Label-free MS imaging from drug discovery to preclinical development.

To fully understand the drug mechanism of action of new chemical entities, pharmacologists need to acquire confident and precise data in pharmacokinetics and in pharmacodynamics and build strong pharmacokinetic/pharmacodynamic relationships. Target engagement in evaluating new chemical entities provides the basis for treatment efficacy. Classical technologies are sometimes limited or inefficient to provide these precise data; however, label-free MS imaging technology is able to provide these molecular features, spatial distributions, quantification and metabolomics data. Important considerations for imaging biological sections are described. Various applications in pharmacology are presented across different therapeutic areas, where MS imaging answers crucial drug discovery and preclinical development needs.

Buchwald reaction as the key step for the synthesis of metabolically more stable analogs of amodiaquine.

Amodiaquine is one of the most active anti-malarial 4-aminoquinoline but its metabolization is believed to generate hepatotoxic derivatives. Previously, we described new analogs of amodiaquine and amopyroquine, in which hydroxyl group was replaced by various amino groups and identified highly potent compounds with lower toxicity. We describe here the synthesis of new analogs that have been modified on their 4'- and 5'-positions in order to reduce their metabolization. A new synthetic strategy was developed using Buchwald coupling reaction as the key step.