329 Proteomic Analysis of Single Cells Isolated from Solid Tumors

Fast-Track proposals will be accepted.

Number of anticipated awards: 4

Budget (total costs):
Phase I: $160,000 for 9 months;
Phase II: $1,000,000 for 2 years

It is strongly suggested that proposals adhere to the above budget amounts and project periods. Proposals with budgets exceeding the above amounts and project periods may not be funded.

The deadline for receipt of all contract proposals submitted in response to this solicitation has expired. It was: November 25, 2013 by 4:30 p.m. ET.

Summary:

The isolation and proteomic mapping of single cells are essential to understanding cancer disease processes at both the molecular and systems levels. In a given tissue, whole cell populations pose the issues of heterogeneity and a lack of synchronicity, which can be overcome with analysis performed at the single cell level. The ability to monitor and analyze signaling events in individual cells would enhance our knowledge of disease biology in cancer cells. Furthermore, protein changes can occur in both graded and binary fashions in response to differing environmental conditions, but bulk measurements obscure such cellular responses. Thus, identifying single cell variation is necessary for understanding how cells exist as autonomously functioning dynamic systems. While imaging techniques currently exist, it is not possible to analyze a whole proteome or differences in protein expression between individual cells as these methods are low-throughput with limited scope. New advances have been made in mapping a single cell’s proteome using flow cytometry, transition element isotopes, and mass cytometers, each of which relies on viable cells. Single cell approaches that can capture living cells from solid tumors in vivo, ex vivo, or from fixed/frozen solid tumor tissues, combined with innovative proteomic engineering technologies and computational analytic tools, will substantially expand the single cell proteomics field and provide novel insights into cancer biology.

Project Goals:

The goal of this contract topic is to advance the field of single cell proteomics through the optimization of single cell isolation from tumors, and the validation and benchmarking of proteomic analytical methods. Single cell approaches that can effectively and reliably isolate single cells, without the use of an artificial construct or overexpression system, from solid tumor tissues in vivo, from fresh solid tumor tissue ex vivo, or from fixed/frozen solid tumor tissues require improvements in yield, ease-of-use, and reproducibility. Current methods for isolating, manipulating, and tracking cells are limited to either bulk techniques that lack single cell accuracy, or manual methods that provide single cell accuracy, but at significantly lower throughputs and repeatability. Additionally, sensitivity of current single cell technologies does not allow for in depth analysis spanning the whole proteome. Therefore, the integration of medium to high throughput single cell acquisition from solid tumors with whole proteome analysis will enhance our biological understanding of post-translational modifications, signaling circuitries, aberrations and other cancer proteome-related information at both the single cell and global levels. Proposals for single cell isolation technologies from circulating tumor cells, hematological non-solid cancers or blood immune cells will not be considered. Contractors will be expected to work with the Clinical Proteomic Technologies for Cancer (CPTC) community (http://proteomics.cancer.gov), as well as stakeholders in the private and public sectors, during development to ensure that appropriate unmet needs in the field are addressed.

Phase I Activities and Expected Deliverables:

• Design the system and identify the interacting components.
• Build cell isolation prototype.
• Test prototype to demonstrate proof-of-concept functionality using solid tumor clinical samples for proteomic analysis applications including, but not limited to, mass spectrometry, flow cytometry, transition element isotope analysis, and mass cytometry.
• If using fresh unfrozen tumors, demonstrate that cell viability following isolation is >90%.
• Establish a protocol to integrate the single cell isolation technique into a proteomic platform such as mass spectrometry.
• Validate above integrated platform by demonstrating enhanced proteomic analysis coverage compared to current technologies such as mass cytometry. Criteria include, but are not limited to, number of proteins detected, and the limit of detection or detectability of low abundant proteins.
• Present findings to an NCI CPTC Evaluation Panel.
• Research should be proposed with quantitative feasibility milestones.

Phase II Activities and Expected Deliverables:

• Demonstrate medium to high-throughput single cell isolation capacity of the prototype and integrate all of the modules in the proteomic platform.
• Test integrated platform operability.
• Benchmark system by comparing and contrasting the competitive advantage of the new prototype over existing products or services using whole tissue analysis.
• Perform requirement and actual use analysis in such a way that it can be translated into a viable commercial opportunity.
• Verify the scope and application of the single cell analysis system and derived data analysis by using a designed cohort of samples with a specified biological question or application.
• Deploy the system in the production environment and correct any errors that are identified in this phase. Add or modify functionality based on updated requirements.