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NIH/NCI 359: Technologies for Differential Isolation of Exosomes and Oncosomes

Fast-Track proposals will not be accepted

Direct to phase II will not be accepted

Number of anticipated awards: 2-3

Budget (total cost per award):

Phase I: $300,000 for 9 months

Phase II: 1,500,000 for 2 years

PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED.

 

Summary

Both normal and cancer cells shed exosomes and other microvessicles into body fluids. Exosomes collected from the blood and other body fluids of patients diagnosed with various cancers were shown to contain tumor suppressors, phosphoproteins, proteases, growth factors, bioactive lipids, mutant oncoproteins, oncogenic transcripts, microRNA, RNA and genomic DNA (gDNA) fragments.  Exosomal trafficking and reciprocal exchange of molecular information among different organs and cell types was reported to contribute to cell-to-cell communication, horizontal cellular transformation, cellular reprogramming, functional alterations, regulation of immune response, and metastasis.  Exosomes collected from cancer patients were reported to perform cell-independent miRNA biogenesis, and promote tumorigenesis by mediating an efficient and rapid silencing of miRNAs to reprogram the transcriptome of cells that they physically interact with. In functional studies, exosomes derived from serum collected from cancer patients were reported to activate normal epithelial cells to form tumors, while exosomes from healthy individuals appear to have anti-tumor characteristics. Since exosomes are continuously released by all tissue and carry molecular signatures and effectors of health and disease, they reflect the dynamic changes taking place in tissue microenvironments throughout the different stages of cancer progression. Of clinical significance is the possibility that exosomes in blood and other body fluids may offer a non-invasive or minimally invasive way to assess cancer initiation, progression, risk, survival and treatment outcomes of cancer.

Exosomes are found in several biofluids including amniotic fluid, breast milk, bronchoalveolar fluid, cerebrospinal fluid, malignant ascites, plasma, saliva and urine, and studies reported differential molecular profiles of extracellular vesicles (EVs) in cancer patients’ sera/plasma from breast, prostate, lung, liver, gastric, esophageal, glioblastoma, Kaposi's sarcoma-associated herpesvirus-associated malignancies, and urine from prostate. In addition, it has been reported that the concentration of exosomes is higher in the blood of cancer patients.  Unlike cell-free circulating nucleic acids (cfCNA), the exosomal cargo is protected by a phospholipid bilayer membrane.  Therefore, the tissue specific biomolecules contained in the exosomes are stable in the body fluids compared to the cfCNAs.  This stability and the possibility to collect serial samples of biofluids non-invasively or minimally invasively, over a period of time, offers an unprecedented opportunity to obtain reproducible time-varying tissue specific genotype and phenotype information in body fluids that resemble dynamic changes taking place during cancer initiation, tumor development and metastasis in tissues.  Molecular profiles of exosomes in archived samples collected in retrospective and prospective studies may further offer valuable information needed to accelerate cancer research and options for clinical care. 

The major bottle neck for using exosomes in cancer research or clinical care is in obtaining enriched preparations of oncosomes from body fluids, where “oncosomes” are defined in this solicitation as exosomes that contain oncogenic cargo and/or unique signatures of the tumor cells from which they emanate.  Existing technologies are based on centrifugation, precipitation/centrifugation or affinity purification, which are labor intensiveand time consuming.  Currently, we do not have effective technologies that can differentially isolate tissue-specific exosomes and tumor-derived oncosomes from the general population of exosomes in archived body fluids.

The purpose of this contract proposal is 1) to support the development of technologies for differential isolation of tissue-specific exosomes and tumor-derived oncosomes from any body fluid(s), and 2) to obtain enriched, distinct preparations useful for downstream comparative molecular profiling or therapeutic use. Applicants must propose to develop an efficient and cost effective platform for complete isolation and separation of exosomes/oncosomes, which are morphologically and functionally intact.

 

Project Goals

The goal of this contract proposal is to accelerate the use of exosomes from body fluids for cancer research and clinical care. It is also intended for developing technology for differential isolation of tissue-specific exosomes and oncosomes in serial collections of archived body fluids to enable assessment of cancer initiation, progression, risk, aggressiveness, prognosis and/or treatment outcomes.  Since exosomes are continuously released from normal, pre-cancerous, tumor, and metastatic tissues, the time-varying genotype and phenotype of exosomes in body fluids may provide a mechanistic understanding of carcinogenesis, tumor initiation, promotion, development, and progression in tissues, and the knowledge gained may lead to better cancer prevention/care/control. Patient-derived exosomes may also serve as targeted drug/antibody delivery systems and immunomodulation agents to yield new precision medicine strategies.

Applicants are required to obtain distinct preparations of exosomes and oncosomes, which originated in a specific tissue/tumor, from routinely collected fresh/archived body fluids. They should demonstrate quality, quantity and reproducibility of isolation and separation using physicochemical and functional studies. The technology platform should be be useful for profiling multiple body fluids from multiple cancer types. The technology should establish automated workflows to reduce human intervention and obtain exosome preparations suitable for research and therapeutic purposes.

To apply for this topic, offerors should:

Have a prototype platform with demonstrated capability for isolating exosomes from complex solutions. Preference will be given for proposals with demonstrated capability for further isolating oncosomes from the general exosome population.

Demonstrate sufficient expertise and necessary resources for robustly characterizing captured exosomes, and verifying persistence of their biological integrity.

 

Phase I Activities and Deliverables

  • Develop a technology for differential isolation of exosomes and oncosomes, which originated in a specific tissue, from body fluid(s) collected from cancer patients (e.g., breast, prostate, colon, lung or brain). The technology must be sufficient for adoption in clinical workflows and therefore demonstrate capability for processing at least 10 mL of clinical fluid specimen in <1 hour.
  • Demonstrate that the technology can obtain distinct preparations of exosomes and oncosomes from the routinely collected fresh/archived body fluids, and yields sufficient quantity for downstream analysis. Specifically, demonstrate sufficient yield of nucleic acids for NGS and proteins for LC-MS/MS
  • Establish automated workflows sufficient to allow for minimal training for new users.
  • Demonstrate that the reproducibility is >90% and yield is >70%
  • Demonstrate collection of >75% intact and undamaged exosomes/oncosomes is using physicochemical methods (Transmission electron microscopy, AFM, dynamic light scattering, immunostaining/immunofluorescence).
  • Benchmark the developed technology against at least 2 current techniques (e.g. centrifugation, density gradient, immunocapture, size-based filtration, etc.) and demonstrate comparable purity and yield from clinically appropriate sample sizes for the specific bodily fluid.
  • Deliver to NCI the SOPs for exosome/oncosome isolation, and the data from physicochemical characterization that demonstrates the quality of the isolated exosomes/oncosomes

 

Phase II Activities and Deliverables

Adapt the technology to multiple body fluids (i.e., stored or freeze thawed) with varying complexity.

Demonstrate that the isolated exosomes/oncosomes are morphologically intact by physicochemical methods (Transmission electron microscopy, AFM, dynamic light scattering, immunostaining/immunofluorescence), and functionally active in in vitro systems (transmission of information from exosomes to cells in culture and/or co-culture). 

Develop a pre-commercial prototype kit/tool/device for the differential isolation of exosomes/oncosomes.

Posted: August 1, 2016