Clara Feider (Presenter)
University of Texas at Austin
Bio: Clara Feider is a graduate student in Prof. Livia Eberlin's group at the University of Texas at Austin.
Authorship: Clara L. Feider (1), Jialing Zhang (1), Suzanne Ledet (2), Spencer Woody (1) Katherine Sebastian (2), , Michael T. Breen (3), Livia S. Eberlin (1)
(1) The University of Texas at Austin, Austin, TX 78712, (2) Seton Medical Center, Austin, TX 78705, (3) Dell Medical School, Austin, TX 78712
Here, we describe the use of ambient ionization mass spectrometry (MS) techniques towards the molecular analysis of endometrial glands and stroma found within both healthy endometrial tissue and endometriosis lesions. Using ambient ionization MS analysis of the lipid and metabolite species in endometriosis lesions, we aim to find molecular ions that are indicative of endometriosis compared to normal endometrium from the same patients. The information gathered in this studied will be used to: 1) evaluate statistically significant alterations between healthy and diseases endometrium to yield insights into endometriosis pathogenesis; and 2) investigate the usefulness of intraoperative ambient MS during conservative endometriosis surgery to improve precision in endometriosis resection, reduce disease recurrence, and ultimately improve patient outcome.
Endometriosis is a debilitating illness that effects approximately 10% of women in their reproductive years, characterized by abnormal and uncontrolled growth of the endometrium tissue outside the woman’s uterus . Women suffering from endometriosis often experience infertility, dysmenorrhea, and abnormal uterine bleeding. However, despite its prevalence and impact on a patient’s quality of life, the causes and pathogenesis of endometriosis are not well understood . Despite many attempts, there are currently no definitive theories of the cause of endometriosis or disease biomarkers . Currently, the most common diagnosis and treatment method for patients is lesion resection through an invasive laparoscopic surgery to remove the suspected lesions from the abdominal cavity, which are then evaluated for endometrial tissue by pathology. However, surgeons must strike a balance between complete resection of endometriosis lesions, which often bear a close resemblance to adjacent normal structures, and conserving healthy tissue as to preserve a woman’s fertility post-surgery. Further, increasing research suggests that microscopic endometriosis lesions that cannot be visualized during traditional laparoscopy may exist on the peritoneal walls of women suffering from the disease.  Presently, there are minimal imaging tools to assist in the differentiation between endometrial and surrounding tissues, resulting in incomplete endometriosis resection and potential disease reoccurrence .
Mass spectrometry (MS) imaging has been widely employed to investigate protein, lipid and metabolite alterations that occur within diseased tissues compared to normal counterparts [6-9]. In particular, ambient ionization MS imaging provides extensive molecular information with minimal sample preparation, as well allowing for acquisition of two-dimensional spatial distribution information of the molecules within the tissues. Through association of 2D molecular images with pathological evaluation of the same tissue sections, disease state of a tissue section is correlated to a molecular profile for tissue classification. As endometriosis presents as unregulated cellular growth in an abnormal location, it is hypothesized that metabolic changes are associated with the dysregulated endometrium cells compared to normal endometrium,[10, 11] which may be detectable by ambient ionization MS.
Here, we employ ambient ionization MS methods to investigate metabolic changes associated with endometriosis and to evaluate the feasibility of these technologies for intraoperative detection and diagnosis of endometriosis. Initially we utilized desorption electrospray ionization (DESI) MS imaging to chemically and spatially characterize endometriosis lesions as well as normal endometrium to compare the metabolite composition of both healthy and diseased endometrial tissue. Using DESI-MS imaging, we were able to isolate endometrial glands and stroma and scar tissue within lesions, which can be as small as 100 μm in size, to compare the molecular ion abundances between these glands and stroma to those found within healthy endometrium. This molecular information was used to characterize the species associated with endometriosis to aid in diagnosis and potentially give insights into disease pathogenesis and potential disease biomarkers. Furthering the study to evaluate MS as a clinical tool towards more complete resection of endometriosis lesions during laparoscopic surgery, we have analyzed the endometriosis lesions that have been pathologically confirmed as endometriosis with the MasSpec Pen, a non-destructive, biocompatible clinical tool we have recently reported for in vivo tissue analysis. We are currently evaluating the effectiveness of the MasSpec Pen in the detection of endometriosis from surrounding structures in the abdomen with the ultimate goal of improving resection efficiency during conservative endometriosis surgery.
Endometriosis lesions and normal endometrium tissue samples were obtained from Seton Medical Center under IRB approved protocol. Fresh samples were flash frozen with liquid N2 and stored in a -80oC freezer until sectioned. Tissue samples were sectioned at 16 µm thick using a CryoStarTM NX50 cryostat (Thermo Fisher Scientific, San Jose, CA). After sectioning, the glass slides were stored in a -80oC freezer. Prior to MSI, the glass slides were dried for ~15 min.
A 2D Omni Spray (Prosolia Inc., Indianapolis, IN) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, San Jose, CA) at 60,000 resolving power at m/z 200 was used for tissue imaging. DESI-MS imaging was performed in the negative ion mode from m/z 100-1500. The histologically compatible solvent system dimethylformamide:acetonitrile (DMF:ACN) 1:3 (v/v)  was used for analysis, at a flow rate of 1.4 µL/min at a spatial resolution of 100μm.
The same tissue sections analyzed by DESI-MSI were stained using standard H&E staining protocol. Pathologic evaluation was performed using light microscopy. Regions of endometrial stroma and glands were noted.
Lipid and metabolite species were identified using high mass accuracy measurements and collision induced dissociation (CID) tandem MS analysis, performed on the Orbitrap Elite at 120,000 resolving power. Lipid fragmentation patterns were compared to literature reports and used in conjunction with data from Lipidmaps database (www.lipidmaps.org) for identification.
2D imaging data analysis
Xcalibur RAW files were converted into images using FireFly data conversion software (Prosolia, Inc. Indianapolis, IN) and then uploaded into the open source imaging software package BioMAP (Novartis).
MasSpec Pen Analysis
PTFE tubing (ID, 1/32 inch; outer diameter, 1/16 inch) was directly inserted into the 3D printed PDMS probe tip for experiments. All MS-Pen experiments were performed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose,CA) with a range of m/z 120 to 1800, using a resolving power of 140,000 at m/z 400. Water was used as the extraction solvent. Location of endometriosis lesion was determined by H&E staining of a tissue section of the same region and analyzed by a pathologist.
DESI-MS imaging has been performed on a set of normal endometrium and endometriosis lesions in the negative ion mode in order to detect the molecular distribution of glycerophospholipids, glycerosphingolipids, and metabolites that are present within the lesions and normal endometrium tissues. To date we have imaged 127 samples, encompassing 25 normal endometrium tissues and 102 endometriosis lesions from 54 patients and are continuing our prospective collection of samples. The same tissue sections that underwent DESI-MS imaging were evaluated by pathology to determine regions of pure endometrial glands and stroma. Visually, mass spectra acquired within tissue samples corresponding to eutopic and ectopic endometrium have observable differences. For example, the relative abundance of a peak at m/z 126.905 corresponding to iodine is drastically increased within the normal endometrium tissue samples compared to the endometriosis lesions. Serval other ions that appear to be upregulated within normal endometrium include m/z 722.518, correlating to a plasmalogen gylcerophosphoethanolamine 36:5, and m/z 861.549, corresponding to PI 36:2. We observed other ions that appear to be more abundant within endometrial glands/stroma from the lesions, including m/z 771.517, corresponding to PG 36:3, and m/z 913.582, correlating to PI 40:4. We are currently evaluating the statistical significance of the observable differences to 1) evaluate if there are statistically significant differences between endometriosis lesions and normal endometrium and 2) determine which molecular ions are the major contributors to distinguishing between the two tissue types.
The MasSpec Pen was used to analyze 18 endometriosis lesions, yielding similar molecular information from that observed with DESI-MS. We plan to continue sample collection and analysis, followed by construction of a molecular classification model for real-time detection of endometriosis using the MasSpec Pen.
Conclusions & Discussion
Here, we apply DESI-MS imaging and the MasSpec Pen to evaluate metabolite changes associated with the endometrium tissue found within endometriosis lesions compared to normal counterparts as well as determine the usefulness of these technologies for accurate detection of endometriosis for future use in laparoscopic surgery. By applying DESI-MS imaging to the analysis of normal endometrium and diseased counterparts, we have observed alterations in the lipid and metabolite profiles between the histologically identical tissues. Characterization of statistically significant molecular ions will be performed and biological hypotheses will be drawn to yield insight into endometriosis pathogenesis and cellular alterations that occur within ectopic endometrium. Additionally we have begun evaluating the potential for in vivo detection of endometriosis lesions using the MasSpec pen, aiming to improve detection of microscopic endometriosis lesions, potentially increasing the probability of full resection during surgery and reducing disease recurrence.
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This work was supported by the Texas Health Catalyst, the AAAS Marion Mason Award for Women in Chemical Sciences, the National Institutes of Health (grant 4R00CA190783-02), and the National Science Foundation (grant CHE-1559839). We thank Marta Sans, Kyana Garza, and Anna Krieger for assistance with experiments.
IP Royalty: no
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