Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Abstract

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis 1 . The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample 2, 3 . Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment.Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease 4 . While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression 5 . The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies [6][7][8] . Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency 9,10 . Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish 8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish. Video LinkThe video component of this article can be found at https://www.jove.com/video/2790/ Protocol 1. DNA microinjection of zebrafish embryos 1. Linearize rag2::cMyc and rag2::GFP 11 DNA constructs by digesting 10μg of each plasmid with NotI at 37°C overnight.2. Phenol:chloroform extract and precipitate the plasmids, and resuspend each in 20μL H 2 O. 3. Determine the...

A Quantitative System for Studying Metastasis Using Transparent Zebrafish

“…We estimate 1 MIC per 119,311 cells (lower bound=1/78,019; upper bound=1/228,233), meaning that this is the frequency of cells within the ZMEL1 population capable of giving rise to measurable distant metastases within 14 days. This is in agreement with other reports indicating that the frequency of cells capable of completing all steps in the metastatic cascade are quite rare (40, 41). These two measures, the μ score and the MIC frequency, are key measures of metastasis in the zebrafish.…”

Clonal Evolution Enhances Leukemia-Propagating Cell Frequency in T Cell Acute Lymphoblastic Leukemia through Akt/mTORC1 Pathway Activation

“…Monoclonality was verified by detection of a single tcr β rearrangement (Blackburn et al, 2012, Table S1) and in some cases by array CGH. Serial passaging of monoclonal T-ALL was accomplished using similar protocols as outlined previously (Blackburn et al, 2011). Kaplan Meier analyses were performed using a Log-rank (Mantel-Cox) test in GraphPad Prism.…”

Danio rerio: Small Fish Making a Big Splash in Leukemia

“…Groups often use transplant to validate their leukemia models, as serial allo-engraftment is generally held to be an indicator of true malignancy, but more elegant allo-transplantations are also reported. For example, leukemia-initiating cell (LIC) frequencies in D. rerio T-ALL have been calculated using limiting-dilution transplants [20, 76, 81, 82]. Still, host immunosuppression by pre-irradiation tempers this assay's biologic relevance.…”

“…Still, host immunosuppression by pre-irradiation tempers this assay's biologic relevance. Several studies shrewdly avoided this issue with syngeneic fish [46 •• , 79, 82, 83]; in one, engraftments were achieved after single cell transplants [79]! This approach has also yielded insight into the transition from pre-leukemic polyclonal expansion to outright neoplasia [46 •• ].…”