Sali Liu

In Vivo Imaging of Tumor-Propagating Cells, Regional Tumor Heterogeneity, and Dynamic Cell Movements in Embryonal Rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is an aggressive pediatric sarcoma of muscle. Here, we show that ERMS-propagating potential is confined to myf5+ cells and can be visualized in live, fluorescent transgenic zebrafish. During early tumor growth, myf5+ ERMS cells reside adjacent normal muscle fibers. By late-stage ERMS, myf5+ cells are reorganized into distinct regions separated from differentiated tumor cells. Time-lapse imaging of late-stage ERMS revealed that myf5+ cells populate newly formed tumor only after seeding by highly migratory myogenin+ ERMS cells. Moreover, myogenin+ ERMS cells can enter the vasculature, whereas myf5+ ERMS-propagating cells do not. Our data suggest that non-tumor-propagating cells likely have important supportive roles in cancer progression and facilitate metastasis.

Notch signaling expands a pre-malignant pool of T-cell acute lymphoblastic leukemia clones without affecting leukemia-propagating cell frequency.

NOTCH1 pathway activation contributes to the pathogenesis of over 60% of T-cell acute lymphoblastic leukemia (T-ALL). While Notch is thought to exert the majority of its effects through transcriptional activation of Myc, it also likely has independent roles in T-ALL malignancy. Here, we utilized a zebrafish transgenic model of T-ALL, where Notch does not induce Myc transcription, to identify a novel Notch gene expression signature that is also found in human T-ALL and is regulated independently of Myc. Cross-species microarray comparisons between zebrafish and mammalian disease identified a common T-ALL gene signature, suggesting that conserved genetic pathways underlie T-ALL development. Functionally, Notch expression induced a significant expansion of pre-leukemic clones; however, a majority of these clones were not fully transformed and could not induce leukemia when transplanted into recipient animals. Limiting-dilution cell transplantation revealed that Notch signaling does not increase the overall frequency of leukemia-propagating cells (LPCs), either alone or in collaboration with Myc. Taken together, these data indicate that a primary role of Notch signaling in T-ALL is to expand a population of pre-malignant thymocytes, of which a subset acquire the necessary mutations to become fully transformed LPCs.

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope.

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis.

Quantifying the frequency of tumor-propagating cells using limiting dilution cell transplantation in syngeneic zebrafish.

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 prognosis1. 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 sample2, 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 disease4. 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 regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. 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 frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 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.

Abstract A08: Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation

This abstract is being presented as a short talk in Session 3: Mechanisms of Resistance. A full abstract is printed in the Proffered Abstracts section (PR06) of the Conference Proceedings. Citation Format: Jessica Blackburn, Sali Liu, Kimberly Dobrinski, Sarah Martinez, Finola Moore, Riadh Lobbardi, David Langenau. Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A08.

Abstract PR06: Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation

Leukemia progression and relapse are driven by molecularly distinct and often-rare cancer cells called leukemia-propagating cells (LPCs). If LPCs are retained following treatment, they will ultimately initiate relapse disease. Despite the substantial number of genetic lesions that have been identified in relapse samples and the contention that these mutations likely modulate response to therapy, acquired mutations that increase the overall frequency of tumor propagating cells following continued clonal evolution have not been reported in any cancer to date. Here, we have developed a transgenic zebrafish model where single fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) cells are transplanted into genetically identical recipient fish and functionally assessed for differences in leukemia propagating cell frequency, growth, and dexamethasone resistance. While subclonal variation was observed within single cells from the same primary leukemia, a subset of clones continued to evolve genetic lesions and epigenetic modifications to enhance growth and overall LPC frequency. A majority of evolved clones acquired activated AKT signaling, which simultaneously increased the number of leukemia propagating cells through activating the mTORC1 pathway, enhanced growth by stabilizing Myc protein levels, and rendered T-ALL cells resistant to dexamethasone, which was reversed by combined treatment with an AKT inhibitor. These results were confirmed using large-scale transgenic epistasis experiments and limiting-dilution cell transplantation studies. In total, our data suggest that diagnosis clones can stochastically acquire mutations necessary to survive treatment and drive relapse even before a patient begins treatment, with acquired mutations being independently selected based on important cancer phenotypes including elevated growth rate and leukemia propagating potential. Moreover, our work also identifies combination therapies that utilize dexamethasone and AKT inhibitor can kill LPCs in a subset of refractory T-ALL. Finally, these are the first studies performed in any model to follow single cell evolution as it relates to relapse, utilizing in excess of 6,000 transplant recipient animals and opening new and exciting avenues of study to uncover genetic pathways that drive cancer malignancy. This abstract is also presented as Poster A08. Citation Format: Jessica Blackburn, Sali Liu, Kimberly Dobrinski, Sarah Martinez, Finola Moore, Riadh Lobbardi, David Langenau. Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR06.

Abstract PR05: Clonal evolution enhances leukemia-propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation

Leukemia progression and relapse are driven by molecularly distinct and often-rare cancer cells called leukemia-propagating cells (LPCs). If LPCs are retained following treatment, they will ultimately initiate relapse disease. Despite the substantial number of genetic lesions that have been identified in relapse samples and the contention that these mutations likely modulate response to therapy, acquired mutations that increase the overall frequency of tumor propagating cells following continued clonal evolution have not been reported in any cancer to date. Here, we have developed a transgenic zebrafish model where single fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) cells are transplanted into genetically identical recipient fish and functionally assessed for differences in leukemia propagating cell frequency, growth, and dexamethasone resistance. While subclonal variation was observed within single cells from the same primary leukemia, a subset of clones continued to evolve genetic lesions and epigenetic modifications to enhance growth and overall LPC frequency. A majority of evolved clones acquired activated AKT signaling, which simultaneously increased the number of leukemia propagating cells through activating the mTORC1 pathway, enhanced growth by stabilizing Myc protein levels, and rendered T-ALL cells resistant to dexamethasone, which was reversed by combined treatment with an AKT inhibitor. These results were confirmed using large-scale transgenic epistasis experiments and limiting-dilution cell transplantation studies. In total, our data suggest that diagnosis clones can stochastically acquire mutations necessary to survive treatment and drive relapse even before a patient begins treatment, with acquired mutations being independently selected based on important cancer phenotypes including elevated growth rate and leukemia propagating potential. Moreover, our work also identifies combination therapies that utilize dexamethasone and AKT inhibitor can kill LPCs in a subset of refractory T-ALL. Finally, these are the first studies performed in any model to follow single cell evolution as it relates to relapse, utilizing in excess of 6,000 transplant recipient animals and opening new and exciting avenues of study to uncover genetic pathways that drive cancer malignancy. This abstract is also presented as Poster B23. Citation Format: Jessica Blackburn, Sali Liu, Sarah Martinez, Kimberly Dobrinski, Finola Moore, Riadh Lobbardi, David Langenau. Clonal evolution enhances leukemia-propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR05.

Abstract 3805: Single cell evolution of AKT pathway activation drives T-cell acute lymphoblastic leukemia relapse.

The aggressive and unpredictable behavior of relapsed T-cell acute lymphoblastic leukemia (T-ALL) presents a major clinical challenge, with >70% of children and >90% of adults unable to survive relapsed disease. Relapsed T-ALL often acquires mutations that are not found in the primary malignancy. It is these new mutations that allow clones to survive treatment and drive relapse growth. In order to identify the genes and pathways responsible for T-ALL relapse, we have developed a transgenic zebrafish model of relapsed T-ALL where single fluorescently-labeled leukemic cells are transplanted into genetically identical recipient fish and functionally assessed for differences in relapse growth. Using serial transplantation of single T-ALL cells and >6,000 recipient animals, we have followed single-cell evolution of T-ALL and identified critical drivers of relapse. These experiments showed that 6 of 49 individual T-ALL cells significantly increased their ability to form relapse over time. Analysis of T-ALL clones pre- and post-evolution showed that AKT pathway activation was correlated with increased relapse potential. Subsequent studies utilizing transgenic zebrafish that over-expressed activated AKT in developing T-ALL demonstrated that AKT signaling increased relapse potential 10-fold. Transgenic epistatic experiments revealed that AKT signaling plays two distinct roles in T-ALL relapse: the AKT/mTORC1 pathway directly enhanced relapse potential, while AKT mediated stabilization of the Myc protein increased T-ALL aggressiveness. Moreover, small molecule inhibition of AKT signaling reduced T-ALL relapse potential in vivo by 25-fold and synergized with Dexamethasone, a common cytotoxic chemotherapy, to significantly enhance cell killing in both zebrafish and human T-ALL. Activation of AKT signaling is associated with poor prognosis and drug resistance in human T-ALL, which, together with our work, suggests that AKT will be a useful molecular target in the treatment of T-ALL. Our experiments have documented the functional heterogeneity of single leukemic cells and identified AKT as a critical driver of T-ALL aggression, relapse formation, and insensitivity to therapy. These are first studies performed in any model to follow single cell evolution as it relates to relapse, opening new and exciting avenues of study to uncover genetic pathways that drive cancer malignancy. Citation Format: Jessica S. Blackburn, Sali Liu, Kimberly Dobrinski, Jayme Ranalli, Sarah Martinez, Charles Lee, David Langenau. Single cell evolution of AKT pathway activation drives T-cell acute lymphoblastic leukemia relapse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3805. doi:10.1158/1538-7445.AM2013-3805

Abstract 4293: Notch expression expands the pre-malignant pool of T-cell acute lymphoblastic leukemia clones without affecting self-renewal

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Over 60% of human T-cell acute lymphoblastic leukemias (T-ALL) harbor NOTCH1 activating mutations, and the cMYC pathway is also commonly activated in this disease. Notch and cMyc have been shown to collaborate in mouse transgenic models of T-ALL, suggesting that these oncogenes function in parallel pathways to enhance tumor growth; however, the exact role of each in T-ALL is unknown. Here, we show that co-expression of Notch and cMyc in zebrafish T cells significantly enhances T-ALL progression compared to cMyc or Notch alone (p<0.001). However, Notch co-expression with cMyc does not enhance proliferation, alter cell cycle kinetics, or modify apoptosis in leukemic cells when compared to T-ALLs expressing cMyc alone. Clonality assays using RT-PCR analyses for T-cell receptor-beta rearrangements indicate that Notch increases the number of T-ALL clones contained within the primary tumor when compared to cMyc expressing leukemais. A large portion of T-ALL clones present in primary Notch and Notch/cMyc leukemias cannot transfer disease into transplanted animals, while all cMyc-alone expressing T-ALLs clones are capable of engraftment and reinitiation of leukemia. Large scale limiting dilution cell transplantation analyses using syngeneic zebrafish demonstrated that primary T-ALLs expressing either Notch or Notch/cMyc have 10-fold less leukemia-initiating frequency when compared to T-ALLs that express only cMyc; however, following serial passaging, both the Notch and Notch/cMyc leukemias exhibit similar leukemia-initiating frequency as cMyc-induced T-ALLs, indicating that Notch signaling does not enhance self-renewal of T-ALL initiating cells. Taken together, our data supports a model where Notch expands a pool of pre-malignant T-ALL clones within the primary tumor, a subset of which acquire additional mutations to confer a fully transformed phenotype. By contrast, cMyc alone is insufficient to increase the overall pool of pre-malignant clones but confers a fully-transformed phenotype to leukemic cells, accounting for the longer latency that likely reflects the acquisition of additional genetic changes in clones. Our data may explain why a subset of relapsed human T-ALLs develop from an underrepresented clone found in the primary leukemia, in that primary human T-ALLs likely have a large pool of pre-malignant clones resulting from NOTCH-pathway activation that are unable to self-renew and thus, cannot give rise to relapsed T-ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4293. doi:10.1158/1538-7445.AM2011-4293