Radiation and Cancer Biology

SS 03 - Biology 1 - Innovative Biologic Approaches to Improve Risk Stratification and Treatment Outcomes

27 - Proteomic and Functional Cellular Response to Exosomes Derived from Patients With Locally Advanced Pancreatic Cancer Undergoing Chemoradiation

Sunday, October 21
2:05 PM - 2:15 PM
Location: Room 008

Proteomic and Functional Cellular Response to Exosomes Derived from Patients With Locally Advanced Pancreatic Cancer Undergoing Chemoradiation
K. C. Cuneo1, M. An2, J. Zhu2, J. Wu2, and D. M. Lubman2; 1Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, 2Department of Surgery, University of Michigan, Ann Arbor, MI

Purpose/Objective(s): The effectiveness of chemoradiation in patients with locally advanced pancreatic cancer (LAPC) is limited by the high incidence of metastatic disease and resistance to systemic therapy. In this study, we analyzed the cellular response to exosomes derived from patients receiving chemoradiation for pancreatic cancer. We hypothesized that exosomes derived from cancer patients would induce functional and proteomic changes in pancreatic cancer cells.

Materials/Methods: After obtaining informed consent, blood samples were collected from 10 patients with LAPC planning to receive chemoradiation therapy. Pooled human serum was used for healthy controls. Exosomes were isolated from serum using ultracentrifugation. Transmission electron microscopy (TEM) and NanoSight analysis were used to quantify exosome number and size distribution. A human pancreatic cancer cell line (PANC-1) was exposed to exosomes derived from cancer patients or healthy controls. Effects on cell migration were tested using transwell chambers and effects on cell proliferation were analyzed using a WST-1 assay with gemcitabine. We then analyzed the proteome of PANC-1 cells before and after exposure to patient derived and control exosomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results: TEM and NanoSight analysis showed a similar number and size distribution of exosomes derived from cancer patients and healthy controls. Exposure of PANC-1 cells to patient derived exosomes increased cell migration by 21.5% compared to healthy controls. Additionally, PANC-1 cells had a 14% increase in proliferation in the presence of cancer exosomes compared to healthy controls. PANC-1 cells exposed to patient exosomes were more resistant to gemcitabine concentrations ranging from 1 µM to 10 µM compared to control exosomes. For our proteomic analysis, a total of 4102 proteins were identified by LC-MS/MS. 35 proteins were up-regulated and 27 proteins were down-regulated in PANC-1 cells after incubation with patient derived exosomes relative to control exosomes. We verified the mass spectrometry results of 3 key proteins CD44 (log2(cancer/control)=1.13, p=0.00026), PPP2R1A (log2(cancer/control)=2.13, p=7.5E-05) and p53 (log2(cancer/control)=-1.023, p=9.5E-06) by Western blot. Ingenuity Pathway Analysis suggests that cancer exosomes participate in the pathways of Remodeling of Epithelial Adherens Junctions and Integrin Linked Kinase in PANC-1 cells to promote cell migration and proliferation.

Conclusion: In this work we show that exosomes derived from patients with locally advanced pancreatic cancer enhance cell migration, proliferation, and resistance to therapy compared to exosomes derived from healthy controls. Proteomic analysis of the treated cells identified several proteins of interest including CD44, PPP2R1A, and p53. Targeting pathways related to these proteins could potentially alter therapeutic resistance and the metastatic potential of cancer cells.

Author Disclosure: K.C. Cuneo: Research Grant; Grant Funding. Service Chief; Ann Arbor Veterans Hospital. M. An: Research Grant; NIH. J. Zhu: Research Grant; NIH. J. Wu: None. D.M. Lubman: Research Grant; NIH.

Kyle Cuneo, MD

University of Michigan

Disclosure:
Employment
University of Michigan: Assistant Professor: Employee

Compensation
Grant Funding: Research Grants

Leadership
Ann Arbor Veterans Hospital: Service Chief

Biography:
Kyle Cuneo, MD is an Associate Professor in the Department of Radiation Oncology at the University of Michigan. His research and clinical focus is on gastrointestinal malignancies. Kyle graduated from Cornell University with a degree in Biological and Environmental Engineering in 2002. He completed medical school and an internship at Vanderbilt Medical Center prior to his Residency at Duke University Medical Center. Kyle has been faculty at the University of Michigan since 2013.

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