High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia

Smith, Alexandra; Raimondi, Aubrey R.; Salthouse, Chris D.; Ignatius, Myron S.; Blackburn, Jessica S.; Mizgirev, I. V.; Storer, Narie Y.; Jong, Jill L. O. de; Chen, Aye Tin Maung; Zhou, Yi; Revskoy, Sergei; Zon, Leonard I.; Langenau, David M.

doi:10.1182/blood-2009-10-246488

Cited by 131 publications

(157 citation statements)

References 28 publications

(55 reference statements)

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“…To identify potential variants in immune loci, we first performed whole-exome sequencing of two different lines of homozygous diploid, clonal golden zebrafish: CG1 and CG2 (35,36). Analysis of our exome data revealed distinctive patterns of haplotypic variation.…”

Section: Resultsmentioning

confidence: 99%

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Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

McConnell

Hernandez

Wcisel

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e. We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/ Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates.comparative genomics | proteasome and TAP evolution | major histocompatibility | MHC class I pathway | CG2 clonal zebrafish

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Section: Resultsmentioning

confidence: 99%

“…CG1 and CG2 represent two homozygous diploid (clonal) zebrafish lines (35,36) derived by in-crossing isogenic offspring after two consecutive rounds of parthenogenesis (71). Each line was cloned from outbred AB zebrafish (72) carrying the golden allele (73).…”

Section: Methodsmentioning

confidence: 99%

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

McConnell

Hernandez

Wcisel

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…However, these strains are not yet commonly used in all laboratories, and some zebrafish cancer models exist only in allogenic fish. Limiting dilution cell transplantations can be performed in wild-type strains that have undergone immune ablation, although the self-renewing cell frequency may be calculated as 10-100 fold higher than if syngeneic zebrafish were used 8 . It is important to note that greater doses of cells should be used for transplants into non-immune matched, irradiated recipients, as some tumor cells will not survive transplant into a foreign microenvironment.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

“…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.…”

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

Blackburn

Liu

Langenau

2011

JoVE

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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...

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Animal Models of Lymphoid Malignancies

Mishra

2023

Precision Cancer Therapies, Volume 1 ‐ Targeting Oncogenic Drivers and Signaling Pathways in Lymphoid Malignancies

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High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia

Cited by 131 publications

References 28 publications

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

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

Animal Models of Lymphoid Malignancies

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