Fast-Track proposals will be accepted.
Direct-to-Phase II will not be accepted.
Number of anticipated awards: 3–
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 5, 2014 by 4:30 PM ET.
A vacutainer blood collection tube is a septum-sealed sterile tube with an internal vacuum that facilitates drawing of blood into the tube. Specialized vacutainers containing additives or stabilizers enable many important clinical measurements: vacutainers containing heparin or sodium citrate for plasma isolation; EDTA-containing vacutainers for isolating blood cells; sodium polyanethol sulfonate for blood culture specimens; acid-citrate-dextrose for blood banking studies; and cell preparation tubes (CPTs) with anticoagulant and a density gradient or gel for isolating peripheral blood mononuclear cells.
Several decades ago, clinical metastasis of solid tumors was linked to blood-borne dissemination of tumor cells in the circulation, and clinical instrumentation is now available to isolate and enumerate these circulating tumor cells (CTCs) in a venous blood draw. One of the first instruments cleared by the FDA for in vitro diagnostic use in enumerating CTCs is the CellSearch™ system from J&J/Veridex, which enumerates CTCs that are positive for a cell surface marker called EpCAM.
CTCs are fragile and tend to degrade within a few hours when collected in standard evacuated blood collection tubes, so Veridex developed a proprietary vacutainer called a CellSave Preservation Tube that stabilizes CTCs up to 96 hours at room temperature, which allows shipment of samples to central reference laboratories for analysis. However, the proprietary chemistry of the CellSave Preservation Tube preserves the CTCs for analysis by fixation, so the cells are no longer viable or proliferative and are not suitable for many downstream applications that depend on the ability of the tumor cells to proliferate (i.e., in vitro culture and/or xenograft development).
In addition to the CellSearch technology, several other CTC isolation platforms are now on the market such as the ApoStream device which separates tumor cells based on their dielectric potential which allow for the capture of viable circulating tumor cells some of which have the capacity to proliferate.
The goal of this SBIR topic is the refinement of existing CTC preparation tubes or new development of vacutainers for commercialization that are capable of preserving the viability of solid tumor-derived cancer cells in venous blood for up to 96 hours of transit. Emphasis should be given to developing conditions that promote the survival of tumor cell populations that retain some degree of proliferative capacity. Such a product will enable derivation of new cell lines and patient-derived xenograft (PDX) models from clinical blood specimens that are more readily available than tumor biopsies.
Emergent needs in medical testing and blood-based cell measurements drive development of vacutainer chemistries that preserve cell viability, and once these clinical measurements become important for medical practice, the new vacutainer chemistry is readily commercialized.
There is a great demand for the development of patient-derived models (PDMs) – both cell lines and xenografts – for a number of clinical translational applications. Importantly, these models will need to reflect both the evolution and heterogeneity of a solid tumor, which implies that multiple longitudinal samples of tumor will be needed over the course of an individual patient's therapies, responses and relapses. Because of high biopsy-associated risk, it is unlikely that tumor biopsies or resections will be able to meet this need, but the low risk of venous blood draws makes circulating cancer cells an attractive alternative for collecting tumor material that likely retains the proliferative ability required to establish cell lines and xenograft tumors.
The key to successful use of circulating tumor cells for establishing PDMs is maintaining cell viability and function (especially proliferative function) during the collection, handling and shipping of the blood specimen to central laboratory sites that have the capability to establish PDMs.
Viability of cancer cells in general depends on stimulation of the survival pathways often shared with the normal stem cell counterpart that are not already activated via mutations and other abnormalities. For example, Clevers and colleagues showed that in vitro survival and proliferation of normal gastrointestinal stem cells into organoids require the addition of cell culture supplements that cover six critical signaling pathways, all of which were similarly altered in selected tumors:
|Supplied Factor||Pathway||Potential Mechanism of Cancer Cell Addiction and/or Dependence|
|EGF||activates RAS/RAF/MEK/ERK signaling||mutant KRAS|
|Noggin||blocks BMP signaling||mutant BMPRIA or SMAD4|
|R-spondin||activates WNT signaling||mutant APC or CTNNB1|
|WNT3A||activates Wnt signaling pathway(s)||abnormal Frizzled signaling|
|Jagged-1 peptide||Notch signaling||mutant Notch/upregulation of Jagged-1|
|Matrigel w/Y-27632||Cell adhesion||Modulated expression of cadherins, selectins, and integrins|
|N-acetyl-cysteine||Protection from oxyradical damage||Induced protective enzymes|
Whereas normal GI stem cells require all of these factors to survive and function in vitro, malignant GI stem cells can harbor mutations and other abnormalities (shown in the table above) that confer factor-independence to one or more of these essential pathways. Each individual carcinoma may harbor a different set of these abnormalities, so each case may require its own specific set of supplied factors for promoting cancer survival in blood during transport. In the absence of genomic information to identify pathways that are factor-independent, multiple pro-survival factors may be needed to maximize viability of different types of tumor specimens during collection, processing and shipment.
The current understanding of normal stem cell survival and of tumor culture growth conditions may identify pro-survival factors that will maximize recovery of circulating tumor cells out of a vacutainer. The analogy may be anatomic, e.g., the cocktail of factors promoting survival of normal GI stem cells or cultured GI tumor cells may promote survival of circulating cancer cells from colorectal carcinomas (or subtypes). The analogy could also be embryologic, e.g., the cocktail that promotes survival of stem cells in endoderm-derived tissues or cultured endodermal tumors may promote survival of circulating tumor cells from malignancies of endoderm-derived tissues.
The short-term goal is identification of the minimum cocktail of survival factors for circulating tumor cells, possibly using knowledge of survival factors of corresponding normal stem cells or cultured tumor cells. In addition to protein factors, other important variables in the cocktail may include extracellular matrix, metabolic substrates (e.g. glucose, glutamine), small molecule metabolites, pH, dissolved gases, and alternative anti-coagulants. It is preferred that general viability of the tumor cells be assessed with the MTT assay and functionality be assessed via a spherogenic assay (other assays may be used with NCI approval). Once recovery, viability, and function are established in the in vitro assays, then NCI will initiate xenograft mouse studies to generate data that further supports the maintenance of tumor cells with proliferative capacity in the presence of the customized tumor cell survival cocktails.
The long-term goal is the adaptation of the survival cocktails to the vacutainer environment either with new configurations or using current CTC prep tubes, including proof of tumor cell recovery, survival, and function in prototype vacutainers containing blood for up to 96 hours of storage. Note that the vacutainer tubes should contain an anti-coagulant with the use of K3EDTA which is recommended by NCI.
Bendas et al (2012) Cancer Cell Adhesion and Metastasis: Selectins, Integrins, and the Inhibitory Potential of Heparins. International Journal of Cell Biology 2012: 1–10
Rodilla et al (2009) Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer. PNAS 106: 6315–6320
Avila et al (2013) Notch signaling in pancreatic cancer: oncogene or tumor suppressor? Trends in Molecular Medicine 5: 320–327
Sato et al (2009) Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459:262–265
Sato and Clevers (2013) Primary Mouse Small Intestinal Epithelial Cell Cultures. Methods in Molecular Biology 945:319–328
Sato and Clevers (2013) Growing Self-Organizing Mini-Guts from a Single Intestinal Stem Cell: Mechanism and Applications. Science 340:1190–1194
Yin et al (2013) Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nature Methods Dec 1. doi: 10.1038/nmeth.2737 [Epub]
Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A, Rimm DL, Wong H, Rodriguez A, Herschkowitz JI, Fan C, Zhang X, He X, Pavlick A, Gutierrez MC, Renshaw L, Larionov AA, Faratian D, Hilsenbeck SG, Perou CM, Lewis MT, Rosen JM, Chang JC. Proc Natl Acad Sci U S A. 2009 Aug 18;106(33)
Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.
Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, Hilsenbeck SG, Pavlick A, Zhang X, Chamness GC, Wong H, Rosen J, Chang JC.J Natl Cancer Inst. 2008 May 7;100(9):672-9
In vitro Analysis of Neurospheres Derived from Glioblastoma Primary Culture: A Novel Methodology Paradigm. Pavon LF, Marti LC, Sibov TT, Malheiros SM, Brandt RA, Cavalheiro S, Gamarra LF.Front Neurol. 2014 Jan 7;4:214.
Prostate Cell line spheroids
Propagation of human prostate cancer stem-like cells occurs through EGFR-mediated ERK activation. Rybak AP, Ingram AJ, Tang D. PLoS One. 2013 Apr 19;8(4)
Colorectal Carcinoma cell line spheroids
NANOG modulates stemness in human colorectal cancer. Zhang J1, Espinoza LA, Kinders RJ, Lawrence SM, Pfister TD, Zhou M, Veenstra TD, Thorgeirsson SS, Jessup JM. Oncogene. 2013 Sep 12;32(37):4397-405.
The offeror will identify the minimum cocktail of survival factors for circulating tumor cells for one type of solid carcinoma malignancy. The offeror may use knowledge of survival factors for stem cells of the normal tissue counterpart and of tumor cell culture conditions. NCI will provide the appropriate cell line.
The cocktail should enable at least 50% recovery and maintain the viability and function of cancer cells spiked into fresh human blood from normal subjects of appropriate blood types for up to 96 hours under typical temperature conditions of transport. The viability, MTT, and functional spherogenic assays are to be performed on the recovered tumor cells after 96 hours.
Activities and deliverables include the following: