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Universal Internal Standard for Reproducible Accurate Quantification of Exosome Protein Markers

Award Number: R01CA258240
ORGANIZATION: NATIONAL CANCER INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 01/14/2022
PERIOD OF PERFORMANCE END DATE: 12/31/2026

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Universal Internal Standard for Reproducible Accurate Quantification of Exosome Protein Markers - Abstract: Pancreatic cancer is currently the third leading cause of cancer related to death in the USA with a 5- year survival rate of <5%. Most potentially curable patients present with unresectable locally advanced disease where the standard of care includes a combination of chemotherapy and radiation therapy. A major challenge in the management of pancreatic cancer is the early assessment of treatment response. Novel markers of early treatment response are critically needed to make better informed decisions regarding the type and sequencing of therapies, given the high toxicity associated with these treatments. In our previous work we were able to identify 20+ exosomal protein markers from the serum of patients with locally advanced pancreatic cancer during therapeutic treatment by using exosome isolation methods and mass spectrometry analysis. However, such analysis for large clinical cohorts was limited by the reproducibility in exosome preparation for accurate quantification of exosomal proteins, which is a significant problem in the exosome omics research field. In the current proposal, we plan to develop the analytics of exosome isolation and characterization with a focus on rigor and reproducibility. We will develop a super-SILAC-based (Stable Isotope Labeling by/with Amino acids in Cell culture) exosome method as a universal internal standard (UIS). In principle, when the super-SILAC- based exosome is spiked into human serum for proteomic analysis it can follow the same experimental workflow as serum exosomes which could maximally reduce variations introduced by sample preparation and LC-MS (Liquid Chromatography-Mass Spec) analysis. Therefore, SILAC-labeled exosomes will be ideal with a dual function for biomarker analysis for reproducible sample preparation and acting as a UIS. Targeted LC-SRM and PRISM-SRM exosome analyses of markers from our prior studies will be used with the UIS to precisely monitor changes in proteins during a course of chemo-radiation therapy. Patients with locally advanced pancreatic cancer will undergo serial blood draws prior to, during, and after treatment with chemotherapy followed by chemo-radiation. We will then perform a targeted proteomics confirmation study based on exosome markers identified in our previous work that have a specific response to a course of chemo-radiation therapy. The marker value changes compared to baseline will be associated with the treatment outcomes (such as progression-free survival and overall survival) in patients. This work will establish the potential use and measurement reproducibility of these exosomal markers for monitoring changes in protein markers during and after treatment. The LC-SRM mass spec-based assay to be used herein has the distinct advantage of being multiplexed for over 20 markers simultaneously. The proposed work contains several novel aspects including: 1) the use of super- SILAC to address irreproducibility in accurate quantification of exosome proteins especially for multi-step exosome preparation and 2) multiplexed targeted LC-SRM and highly sensitive PRISM-SRM assays for biomarker analysis in patient serum exosomes.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $389,719 )
2026	2026	REGENTS OF THE UNIVERSITY OF MICHIGAN	1109 GEDDES AVE STE 3300	ANN ARBOR	MI	48109	WASHTENAW	USA	Cancer Detection and Diagnosis Research	000	5	12/22/2025	NON-COMPETING CONTINUATION	$389,719
														Subtotal = $389,719

Issue Date FY: 2025 ( Subtotal = $433,021 )
2025	2025	REGENTS OF THE UNIVERSITY OF MICHIGAN	1109 GEDDES AVE STE 3300	ANN ARBOR	MI	48109	WASHTENAW	USA	Cancer Detection and Diagnosis Research	001	4	6/6/2025	NON-COMPETING CONTINUATION	$43,302
2025	2025	REGENTS OF THE UNIVERSITY OF MICHIGAN	1109 GEDDES AVE, SUITE 3300	ANN ARBOR	MI	48109	WASHTENAW	USA	Cancer Detection and Diagnosis Research	000	4	11/21/2024	NON-COMPETING CONTINUATION	$389,719
														Subtotal = $433,021

Issue Date FY: 2024 ( Subtotal = $448,610 )
2024	2024	REGENTS OF THE UNIVERSITY OF MICHIGAN	1109 GEDDES AVE, SUITE 3300	ANN ARBOR	MI	48109	WASHTENAW	USA	Cancer Detection and Diagnosis Research	000	3	1/12/2024	NON-COMPETING CONTINUATION	$424,999
2024	2024	REGENTS OF THE UNIVERSITY OF MICHIGAN	1109 GEDDES AVE, SUITE 3300	ANN ARBOR	MI	48109	WASHTENAW	USA	Cancer Detection and Diagnosis Research	001	3	4/16/2024	NON-COMPETING CONTINUATION	$23,611
														Subtotal = $448,610

Issue Date FY: 2023 ( Subtotal = $468,551 )
2023	2023	REGENTS OF THE UNIVERSITY OF MICHIGAN	1109 GEDDES AVE, SUITE 3300	ANN ARBOR	MI	48109	WASHTENAW	USA	Cancer Detection and Diagnosis Research	001	2	7/6/2023	NON-COMPETING CONTINUATION	$0
2023	2023	REGENTS OF THE UNIVERSITY OF MICHIGAN	503 THOMPSON STREET	ANN ARBOR	MI	48109	WASHTENAW	USA	Cancer Detection and Diagnosis Research	000	2	1/31/2023	NON-COMPETING CONTINUATION	$468,551
														Subtotal = $468,551

Issue Date FY: 2022 ( Subtotal = $466,913 )
2022	2022	REGENTS OF THE UNIVERSITY OF MICHIGAN	503 THOMPSON ST	ANN ARBOR	MI	48109	WASHTENAW	USA	Cancer Detection and Diagnosis Research	000	1	1/14/2022	NEW	$466,913
														Subtotal = $466,913

Grand Total All Awards = $2,206,814

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Universal Internal Standard for Reproducible Accurate Quantification of Exosome Protein Markers

Award Number: R01CA258240

ORGANIZATION: NATIONAL CANCER INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 01/14/2022

PERIOD OF PERFORMANCE END DATE: 12/31/2026

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