Keely Pierzchalski (Presenter)
Maastricht University M4I
Bio: 2006: Bachelor¡¦s of Science in Medical Technology from University of Delaware 2006: ASCP Board Certification #223119 2006-2007: Technologist in Clinical Microbiology Laboratory, Virginia Commonwealth University Health System (VCUHS), Richmond, VA, USA 2007-2009: Technologist in Clinical Immunology and Clinical Gastroenterology Laboratories, AI DuPont Hospital for Children, Wilmington, DE, USA 2009-2015: Doctor of Philosophy in Pharmaceutical Science at University of Maryland, Baltimore, Founding Campus 2015-Present: Post-doctoral researcher at Maastricht University, M4I- Division of Imaging Mass Spectrometry
Authorship: KA Pierzchalski (1), J Missbach-Guentner (2) (3), D Pinkert-Leetsch (2) (3), F Alves (2) (3), and RMA Heeren (1)
[1] Maastricht University, M4I- Division of Imaging Mass Spectrometry, Maastricht, Netherlands; [2] Uni. Med. Cent. Goettingin, Dept. Haematology and Medical Oncology, Gottingen, Germany; [3] Uni. Med. Cent. Goettingin Institute of Diagnostic and Interventional Radiology, Gottingen, Germany
| Short Abstract Pancreatic cancer is aggressive and difficult to treat with a 5 year survival rate of just 1-16% respective to stage at diagnosis. Mass spectrometry imaging (MSI) is a powerful analytical tool to investigate pancreatic adenocarcinoma (PDAC) tumors in order to characterize the invasive front in the human orthotopic PDAC tumor models, differentiate stroma reaction, and grades of differentiation of primary PDACs, depending on the model. Based on macroscopic and microscopic annotations of the orthotopic PDAC xenograft tumors, peptide profiles were characterized to compare differences between the tumors, non-tumor tissue, tumor margins, invasive regions and metastatic lesions. This information may provide clinically relevant markers for tumor classification, tumor margin assessment and/or therapeutic response prediction. |
Long Abstract
Introduction: Pancreatic cancer is aggressive and difficult to treat with a 5 year survival rate of just 1-16% respective to stage at diagnosis. [1] Currently, resection is the only curative treatment possibility; however, only 20% of diagnoses are eligible for resection. Of resected tumors, 80% have recurrence with metastasis. [1] Understanding molecular compositions of pancreatic tumor cells in conjunction with the molecular spatial information will provide additional knowledge for diagnostic, therapeutic and prognostic use. Mass spectrometry imaging (MSI) is a powerful analytical tool to investigate pancreatic adenocarcinoma (PDAC) in order to characterize the invasive front in the human orthotopic PDAC tumor models, differentiate stroma reaction, and grades of differentiation of primary PDACs, depending on the model. [2] Paraffin embedded orthotopic PDAC tumors with adjacent organ invasion are being analyzed for peptide profiles in order to perform these characterizations with conservation of the peptide species spatial orientation within the tissues and cells.
Methods: Three different PDAC mouse models with distinct growth and metastatic behavior are established by orthotopic implantation of well characterized (1 x 10^6) human pancreatic tumor cells into the head of the pancreas of athymic male nude mice. The BxPC-3 and PancTu1 cell line was chosen as being characterized as moderately differentiated- and Capan-1 as a well differentiated cell line.
Orthotopic transplantation into the head of the pancreas of all three human PDAC cell lines resulted in local tumor growth and tumor invasion into the adjacent organs with frequent involvement into the duodenum and stomach, and with metastatic spread to liver. However, depending on the cell line implanted, tumor growth and dissemination pattern varied in each PDAC mouse model. As PDAC tumors are hardly detectable by visual inspection, the time for the sacrifice defined as time of development after cell transplantation, was chosen based on the presence of typical symptoms of PDAC such as weight loss of more than 20% and jaundice.
From all tumor models, paraffin sections of primary tumor with adjacent organs such as duodenum and stomach, as well as metastasis on the liver are available for MS spectroscopy. Therefore, we use Mass Spectrometry Imaging (MSI) to assess the primary tumor, the invasion front and metastases within distant organs.
Preparation for peptide analysis of paraffin sections for MSI include numerous steps to remove the paraffin (2 x 5 minute wash in xylene), remove lipid/metabolite species (ethanol/chloroform wash gradients), expose proteins for digest with antigen retrieval (Tris buffer, pH 9.0, 95 C, 20-30 minutes), thorough rinsing with diH2O to remove interfering salts, trypsin digest overnight at 37 C, and CHCA matrix application with recrystallization for MSI analysis on the RapifleX MALDI tissuetyper (Bruker, Bremen, Germany).
Results: Eight PDAC tumors used for the present pilot study were initially characterized for infiltration pattern and metastatic spread. In the PancTu1 tumor model, four tumors demonstrated invasion into the stomach and/ or duodenum and 75% of the mice showed additional metastasis into the liver. Two moderately differentiated BxPC-3 tumors had invasion into and completely adhered to the stomach. Two Capan-1 primary tumors did not demonstrate adjacent organ invasion, however, the liver presents with metastatic lesions. Histological examination showed that the PancTu1 and BxPC3 primary tumors grow in distinct tumor nodules, surrounded by stromal compartment, while the Capan1 tumor was organized in duct like structures with broad lumina. Because the characterization of these tumors in this pilot study were confirmed macroscopically and microscopically, further molecular comparisons by MSI were performed to further characterize the non-tumor, tumor, tumor margins, invasive regions and metastatic lesions.
Peptides are analyzed in positive ion, reflectron mode over the mass range of 600-4000 m/z, with resolution of 100 µm. FlexImaging and SCiLs software (Bruker, Bremen, Germany) are used to generate ion-based images, co-register MS images with H&E stains and peak analysis. Statistical analysis is performed with SCiLs software and in-house statistical software including PCA and hierarchical cluster analyses to identify peptide peak specificity in non-tumor, tumor, tumor margins, invasive regions and metastatic lesions. Co-registered H&E and ion generated images are assessed to confirm peptide species localization to cellular and morphological features.
Conclusions: Based on macroscopic and microscopic annotations of the orthotopic PDAC xenograft tumors, peptide profiles were characterized to compare differences between the tumors, non-tumor tissue, tumor margins, invasive regions and metastatic lesions. Further identification of the proteins from which the peptides originate may provide clinically relevant information; such as tumor classification, tumor margin assessment and/or therapeutic response prediction.
References & Acknowledgements:
1. American Cancer Society
2. RA Cardone, M Raffaella Greco, K Zeeberg, A Zaccagnino, M Saccomano, A Bellizzi, P Bruns, M Menga, C Pilarsky, A Schwab, F Alves, H Kalthoff, V Casavola, and SJ Reshkin. (2015) A Novel NHE1-Centered Signaling Cassette Drives Epidermal Growth Factor Receptor¡VDependent Pancreatic Tumor Metastasis and Is a Target for Combination Therapy. Neoplasia. Feb; 17(2): 155¡V166.
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