BACKGROUND. Mammography surveillance protocols after breast cancer treatment vary widely. Some practices recommend performing diagnostic mammography for a certain number of years or indefinitely, whereas others recommend returning immediately to screening. OBJECTIVE. This study's objective was to determine performance metrics of screening digital breast tomosynthesis (DBT) in patients who resume screening mammography immediately after breast cancer treatment, based on the number of years since the breast cancer diagnosis. METHODS. This retrospective study included screening DBT examinations performed from January 2013 to June 2019 in patients who resumed screening mammography immediately after a prior breast cancer diagnosis. Multivariable logistic regression models with generalized estimating equations were used to evaluate associations between screening performance metrics and years since the prior breast cancer diagnosis, controlling for age, race and ethnicity, breast density, presence of a prior screening mammogram, and interpreting radiologist. RESULTS. The study included 8090 patients (mean age, 65 +/- 11 [SD] years) with a prior breast cancer diagnosis who underwent 30,812 screening DBT examinations during the study period. The cancer detection rate (CDR) was 8.6 per 1000 examinations (265/30,812), abnormal interpretation rate (AIR) was 5.7% (1750/30,812), PPV1 was 15.1% (265/1750), sensitivity was 80.3% (265/330), specificity was 95.1% (28,997/30,482), and false-negative rate was 2.1 per 1000 examinations (65/30,812). CDR showed a significant independent positive association with years since breast cancer diagnosis (adjusted OR, 1.03; 95% CI, 1.01-1.05; p <.001), being lowest more than 2 to up to 3 years after diagnosis (4.9 per 1000 examinations) and highest more than 8 to up to 9 years after diagnosis (11.2 per 1000 examinations). AIR showed a significant independent negative association with years since breast cancer diagnosis (adjusted OR, 0.99; 95% CI, 0.98-1.00; p =.01), being highest 1 year or less after diagnosis (7.5%) and lowest more than 5 to up to 6 years after diagnosis (5.0%). CONCLUSION. Among 8090 patients with a prior breast cancer diagnosis, even though the AIR was higher during the year after diagnosis compared with subsequent years, the AIR remained acceptably low (< 10%) in all years. CLINICAL IMPACT. These results support the study institution's mammographic surveillance protocol for patients with a prior breast cancer diagnosis of returning immediately to DBT screening.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7–7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells. Langenau, Pinello and colleagues identify tumor-propagating stem cells in rhabdomyosarcoma that sustain tumorigenesis through integrated single-cell and functional characterization of patient-derived samples and preclinical models in vivo.

T cell immunotherapies have revolutionized treatment for a subset of cancers. Yet, a major hurdle has been the lack of facile and predicative preclinical animal models that permit dynamic visualization of T cell immune responses at single-cell resolution in vivo. Here, optically clear immunocompromised zebrafish were engrafted with fluorescent-labeled human cancers along with chimeric antigen receptor T (CAR T) cells, bispecific T cell engagers (BiTEs), and antibody peptide epitope conjugates (APECs), allowing real-time single-cell visualization of T cell-based immunotherapies in vivo. This work uncovered important differences in the kinetics of T cell infiltration, tumor cell engagement, and killing between these immunotherapies and established early endpoint analysis to predict therapy responses. We also established EGFR-targeted immunotherapies as a powerful approach to kill rhabdomyosarcoma muscle cancers, providing strong preclinical rationale for assessing a wider array of T cell immunotherapies in this disease.

T cell immunotherapies have revolutionized treatment for a subset of cancers. Yet, a major hurdle has been the lack of facile and predicative preclinical animal models that permit dynamic visualization of T cell immune responses at single-cell resolution in vivo. Here, optically clear immunocompromised zebrafish were engrafted with fluorescent-labeled human cancers along with chimeric antigen receptor T (CAR T) cells, bispecific T cell engagers (BiTEs), and antibody peptide epitope conjugates (APECs), allowing real-time single-cell visualization of T cell–based immunotherapies in vivo. This work uncovered important differences in the kinetics of T cell infiltration, tumor cell engagement, and killing between these immunotherapies and established early endpoint analysis to predict therapy responses. We also established EGFR-targeted immunotherapies as a powerful approach to kill rhabdomyosarcoma muscle cancers, providing strong preclinical rationale for assessing a wider array of T cell immunotherapies in this disease.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and is comprised of two major molecular subtypes. Despite sharing features with skeletal muscle, the conservation of underlying cellular hierarchy with human muscle development and the identification of molecularly-defined tumor-propagating cells have not been reported. Using single-cell RNA sequencing of patient-derived RMS, DNA-barcode cell fate mapping, and antibody enrichment and functional stem cell assays using in vitro culture and mouse xenografts, we have uncovered tumor cell hierarchies in Fusion-negative (FN-) RMS that are shared with normal human muscle development. We also identified common developmental stages at which tumor cells become arrested. FN-RMS resemble early muscle found in embryonic and larval development, while fusion-positive (FP-)RMS express a highly specific developmental gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. FP-RMS also have neural-pathway enriched cell states, suggesting less-rigid adherence to muscle development hierarchies in this disease. Finally, we identify a molecularly-defined tumor-propagating cell in FN-RMS that shares remarkable similarity to the newly described bi-potent, muscle mesenchyme stem/progenitor cell that makes both muscle and osteogenic cells.

Zebrafish are an ideal cell transplantation model. They are highly fecund, optically clear and an excellent platform for preclinical drug discovery studies. Traditionally, xenotransplantation has been carried out using larval zebrafish that have not yet developed adaptive immunity. Larval engraftment is a powerful short-term transplant platform amenable to high-throughput drug screening studies, yet animals eventually reject tumors and cannot be raised at 37 °C. To address these limitations, we have recently developed adult casper-strain prkdc−/−, il2rgα−/− immunocompromised zebrafish that robustly engraft human cancer cells for in excess of 28 d. Because the adult zebrafish can be administered drugs by oral gavage or i.p. injection, our model is suitable for achieving accurate, preclinical drug dosing. Our platform also allows facile visualization of drug effects in vivo at single-cell resolution over days. Here, we describe the procedures for xenograft cell transplantation into the prkdc−/−, il2rgα−/− model, including refined husbandry protocols for optimal growth and rearing of immunosuppressed zebrafish at 37 °C; optimized intraperitoneal and periocular muscle cell transplantation; and epifluorescence and confocal imaging approaches to visualize the effects of administering clinically relevant drug dosing at single-cell resolution in vivo. After identification of adult homozygous animals, this procedure takes 35 d to complete. 7 days are required to acclimate adult fish to 37 °C, and 28 d are required for engraftment studies. Our protocol provides a comprehensive guide for using immunocompromised zebrafish for xenograft cell transplantation and credentials the model as a new preclinical drug discovery platform. This protocol describes how to engraft human cancer cells in immunocompromised adult zebrafish. The fish are first adapted to 37 °C, followed by intraperitoneal or periocular muscle transplantation of xenograft cells and fluorescence imaging.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes and is largely driven by the NOTCH/MYC pathway. Yet, additional oncogenic drivers are required for transformation. Here, we identify protein tyrosine phosphatase type 4 A3 (PRL3) as a collaborating oncogenic driver in T-ALL. PRL3 is expressed in a large fraction of primary human T-ALLs and is commonly co-amplified with MYC. PRL3 also synergized with MYC to initiate early-onset ALL in transgenic zebrafish and was required for human T-ALL growth and maintenance. Mass-spectrometry phosphoproteomic analysis and mechanistic studies uncovered that PRL3 suppresses downstream T-cell phosphorylation signaling pathways, including those modulated by VAV1, and subsequently suppresses apoptosis in leukemia cells. Taken together, our studies have identified new roles for PRL3 as a collaborating oncogenic driver in human T-ALL and suggest that therapeutic targeting of the PRL3 phosphatase will likely be a useful treatment strategy for T-ALL.

Cancer xenograft engraftment studies using immune-deficient mice are indispensable for preclinical drug discovery and are required for IND filings that lead to clinical trials. While immune-deficient mice robustly engraft a wide variety of human cancers, high-resolution intravital imaging of transplanted cells is tedious and its high husbandry costs often limit the scale of experiments. In contrast, zebrafish are an ideal cell transplantation model. They are highly fecund; optically clear, permitting dynamic single-cell imaging of fluorescent cancer cells; and are an excellent platform for high-throughput, large-scale studies. We have recently generated two optically clear prkdc-/-, il2rga-/- and rag2-/-, il2rga-/- immune-compromised zebrafish models that lack T-, B-, and NK-cells. These immune-deficient animals can be grown at 37°C and robustly engraft a variety of human cancers including patient-derived xenografts for >30 days. Importantly, tumors grown in immune-deficient zebrafish and mice are indistinguishable in terms of morphology, proliferation rates, apoptosis, and response to therapy. Engraftment of human cells into the superficial periocular muscle also allowed high-resolution, single-cell imaging using conventional confocal microscopy. Using this approach, we performed photoconversion cell lineage tracing of human rhabdomyosarcoma (RMS), a pediatric cancer of the muscle, and identified mutually exclusive migratory and proliferative cell states. We also demonstrated the preclinical efficacy of combination therapy involving olaparib (PARP-inhibitor) and temozolomide (DNA-damaging agent), including the dynamic visualization of therapeutic responses at single-cell resolution through use of the four-color FUCCI cell cycle fluorescent reporter and serial confocal imaging over days. Importantly, this same drug combination also exhibited remarkable efficacy in studies performed in NSG mice and is now moving forward for clinical evaluation in RMS patients. Finally, recent work has focused on assessing the efficacy of immunotherapies in curbing tumor growth, including assessing CAR-T cell and bispecific T-cell engager antibodies (BITES) in vivo. In both of these platforms, dynamic live cell imaging and automated 3D modeling allowed quantification of migratory potential of T cells into the tumor, dynamic remodeling of T-cell morphology changes following interaction with tumor cells, and real-time visualization of T cell-mediated cancer cell killing using fluorescent caspase reporters. In total, our studies have credentialed the immune-deficient zebrafish as a new platform for preclinical drug studies and provides novel technologies that utilize real-time, single-cell imaging for early endpoint analysis. These models are likely to be particularly useful for discovery of immunomodulatory antibodies and immunomedicines in the future. Citation Format: Chuan Yan, Qiqi Yang, Daniel Do, Dalton Brunson, John Iafrate, John Rawls, David M. Langenau. Dynamic single-cell imaging of human cancer growth and therapy responses following engraftment into immunodeficient zebrafish [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR12.

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

Immune compromised mice are an invaluable model for xenograft cell transplantation studies. To date, engraftment into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice has become the gold standard for accessing cell growth, differentiation, pluripotency of stem cell populations, and therapy responses in human cancers [[1]Day C.P. Merlino G. Van Dyke T. Preclinical mouse cancer models: a maze of opportunities and challenges.Cell. 2015; 163: 39-53Summary Full Text Full Text PDF PubMed Scopus (370) Google Scholar] (Fig. 1). However, these murine models are expensive and single cell imaging of engrafted cells requires complex imaging techniques including surgical window implantation into engrafted mice [[1]Day C.P. Merlino G. Van Dyke T. Preclinical mouse cancer models: a maze of opportunities and challenges.Cell. 2015; 163: 39-53Summary Full Text Full Text PDF PubMed Scopus (370) Google Scholar]. By contrast, zebrafish have many attributes that make it an ideal cell transplantation model including high fecundity, low cost and optical clarity (Fig. 1). Current zebrafish xenotransplantation studies have predominantly been carried out using larval recipients, as the adaptive immune system only becomes fully functional by three-weeks post fertilization [[2]Fior R. Povoa V. Mendes R.V. Carvalho T. Gomes A. Figueiredo N. et al.Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts.Proc Natl Acad Sci U S A. 2017; 114: 8234-8243Crossref Scopus (175) Google Scholar]. Despite many important findings coming from cancer xenograft studies using larval zebrafish including elegant pre-clinical modeling and assessing patient responses to therapy [[2]Fior R. Povoa V. Mendes R.V. Carvalho T. Gomes A. Figueiredo N. et al.Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts.Proc Natl Acad Sci U S A. 2017; 114: 8234-8243Crossref Scopus (175) Google Scholar], they are necessarily limited by the numbers of cells that can be engrafted and a short experimental time window that is usually confined to 5–7 days (Fig. 1). In addition, larval zebrafish engraftment studies are carried out at 34 °C–35 °C; yet, many human tumor cells do not grow well at these non-physiological temperatures and most do not form tumor masses akin to those found in xenografted mice or primary human tumors. Finally, because of their small body size and ability to only deliver drugs using submersion therapy, achieving accurate drug dosing and assessing pharmacokinetics is impossible using larval xenografts. To address the limitations of short-term larval engraftment studies, the community has created an array of partially immune compromised strains of zebrafish that robustly engraft allogeneic tissues [3Moore J.C. Tang Q. Yordan N.T. Moore F.E. Garcia E.G. Lobbardi R. et al.Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.J Exp Med. 2016; 213: 2575-2589Crossref PubMed Scopus (47) Google Scholar, 4Tang Q. Abdelfattah N.S. Blackburn J.S. Moore J.C. Martinez S.A. Moore F.E. et al.Optimized cell transplantation using adult rag2 mutant zebrafish.Nat Methods. 2014; 11: 821-824Crossref PubMed Scopus (99) Google Scholar, 5Hess I. Iwanami N. Schorpp M. Boehm T. Zebrafish model for allogeneic hematopoietic cell transplantation not requiring preconditioning.Proc Natl Acad Sci U S A. 2013; 110: 4327-4332Crossref PubMed Scopus (27) Google Scholar, 6Wienholds E. Schulte-Merker S. Walderich B. Plasterk R.H. Target-selected inactivation of the zebrafish rag1 gene.Science (New York, NY). 2002; 297: 99-102Crossref Scopus (345) Google Scholar]. Building on these successes, an optically-clear, homozygous compound mutant prkdcD3612fs, il2rgaY91fs (prkdc−/−, il2rga−/−) casper-strain zebrafish has been developed that lack T, B and natural killer (NK) cells [[7]Yan C. Brunson D.C. Tang Q. Do D. Iftimia N.A. Moore J.C. et al.Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish.Cell. 2019; 177: 1903-1914Summary Full Text Full Text PDF PubMed Scopus (132) Google Scholar] (Fig. 1). These immune deficient animals can survive at 37 °C and robustly engraft a variety of fluorescently-labeled human cancers for in excess of 28 days. Remarkably, the growth kinetics, histology, cell proliferation and apoptotic rates are largely similar when compared to the same tumor engrafted into NSG mice. Yan et al. went on to identify a drug combination, olaparib PARP-inhibitor and the DNA damaging agent temozolomide, that curbed growth of pediatric rhabdomyosarcoma muscle cancers. Moreover, optical clarity of the prkdc−/−, il2rga−/− casper-strain zebrafish allowed dynamic visualization of therapy responses in vivo using clinically relevant dosing using oral gavage and the four-color FUCCI4-cell cycle reporter. Importantly, this work also confirmed that this same drug combination elicited potent anti-tumor responses in mouse xenografts and had the similiar pharmacokinetics as observed in mouse and human [[7]Yan C. Brunson D.C. Tang Q. Do D. Iftimia N.A. Moore J.C. et al.Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish.Cell. 2019; 177: 1903-1914Summary Full Text Full Text PDF PubMed Scopus (132) Google Scholar]. Based on these results, olaparib and temozolomide combination is soon to be tested in a phase II clinical trial, providing the first example of clinical translation for cancer therapy originating from zebrafish xenograft studies. Achieving robust, long-term engraftment of human cancer cells into adult immune compromised zebrafish is an important step forward in preclinical animal modeling. The zebrafish offers many complementing traits to their murine counterparts. First, the optical clarity of casper-strain immune compromised zebrafish is ideal for high-resolution imaging of different cancer cell processes. For example, by using conventional confocal microscopy and a one-step anesthesia procedure involving submerging the animal in tricaine-infused water, we were able to carry out photoconversion cell lineage-tracing of single engrafted tumor cells with an imaging depth of up to 300 μm, in an average of 5 min per transplant animal. Secondly, engrafting patient-derived cancer xenografts (PDXs) into the prkdc−/−, il2rga−/− model is particularly exciting [[7]Yan C. Brunson D.C. Tang Q. Do D. Iftimia N.A. Moore J.C. et al.Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish.Cell. 2019; 177: 1903-1914Summary Full Text Full Text PDF PubMed Scopus (132) Google Scholar]. The eventual hope is that engrafting a patient's tumor into large cohorts of animals will permit testing of a wide array of clinically available drugs, pairing responses in zebrafish avatars with clinical decision-making that stratifies patients into the most suitable treatment for their tumor. Finally, the adult immunocompromised zebrafish model also has the potential to transform pre-clinical animal modeling and drug discovery by administering drugs orally in a clinically achievable manner, increasing the throughput of in vivo screening, and providing faster imaging endpoints that capitalize on the ability to assess drug affects in real-time and at single cell resolution (Fig. 1). Despite the recent successes in engrafting human cancers into immune-deficient zebrafish, the current prkdc−/−, il2rga−/− model is far from perfect. Engraftment efficiency of most human cancer types ranged from 50% to 90%, with some tumor types never engrafting into the model [[7]Yan C. Brunson D.C. Tang Q. Do D. Iftimia N.A. Moore J.C. et al.Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish.Cell. 2019; 177: 1903-1914Summary Full Text Full Text PDF PubMed Scopus (132) Google Scholar]. Rejection in a subset of recipient animals is likely due to several reasons. First, like immune compromised scid mice, the prkdc mutant zebrafish develop "leakiness" over time, resulting in the retention of small populations of residual B cells [[3]Moore J.C. Tang Q. Yordan N.T. Moore F.E. Garcia E.G. Lobbardi R. et al.Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.J Exp Med. 2016; 213: 2575-2589Crossref PubMed Scopus (47) Google Scholar,[8]Tang Q. Iyer S. Lobbardi R. Moore J.C. Chen H. Lareau C. et al.Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.J Exp Med. 2017; 214: 2875-2887Crossref PubMed Scopus (86) Google Scholar]. Because, a subset of teleost B cells have phagocytic activity [[9]Li J. Barreda D.R. Zhang Y.A. Boshra H. Gelman A.E. Lapatra S. et al.B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities.Nat Immunol. 2006; 7: 1116-1124Crossref PubMed Scopus (376) Google Scholar], it is likely that B cell retention may directly impact tumor cell killing. Second, zebrafish have two distinct populations of NK cells [[8]Tang Q. Iyer S. Lobbardi R. Moore J.C. Chen H. Lareau C. et al.Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.J Exp Med. 2017; 214: 2875-2887Crossref PubMed Scopus (86) Google Scholar]. Importantly, the NK-lysin-expressing subset are retained in prkdc−/−, il2rga−/− zebrafish and likely have the ability to produce cytotoxic, anti-microbial peptides that might account for rejection of transplanted cells [[8]Tang Q. Iyer S. Lobbardi R. Moore J.C. Chen H. Lareau C. et al.Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.J Exp Med. 2017; 214: 2875-2887Crossref PubMed Scopus (86) Google Scholar]. Finally, current engraftment studies into the prkdc−/−, il2rga−/− zebrafish require pre-treating fish with clodronate liposomes to deplete macrophages [[7]Yan C. Brunson D.C. Tang Q. Do D. Iftimia N.A. Moore J.C. et al.Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish.Cell. 2019; 177: 1903-1914Summary Full Text Full Text PDF PubMed Scopus (132) Google Scholar]. It is possible that rejection occurs only in those animals with incomplete clearing of macrophages. Clearly, new immune compromised zebrafish models with mutations that fully deplete T, B, macrophages, and all NK cell subsets will likely provide superior engraftment models in the future. With the established prkdc−/−, il2rga−/− zebrafish and the further development of new immunodeficient zebrafish models, we envision the widespread application of immune compromised zebrafish for the study of cancer biology, stem cell and regenerative biology, and assessing therapy responses in vivo. Future studies will likely see the engraftment of human embryonic stem cells and induced pluripotent stem cells (iPSCs). When paired with the large-scale drug discovery platforms available in the zebrafish model, it is also likely that high throughput screens will identify novel factors that influence self-renewal and lineage specification directly within live animals. New transgenic approaches will also likely be complexed with available immune-deficient zebrafish, providing the ability to express human cytokines that support the growth of human CD34+ cord blood cells and peripheral mononuclear blood cells (PMBCs). These humanized models would provide powerful tools to visualize human blood cell development and witness stem cell self-renewal divisions in vivo, akin to elegant studies already available to the allogeneic zebrafish model [[10]Tamplin O.J. Durand E.M. Carr L.A. Childs S.J. Hagedorn E.J. Li P. et al.Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.Cell. 2015; 160: 241-252Summary Full Text Full Text PDF PubMed Scopus (224) Google Scholar]. Moreover, creating "humanized" zebrafish would also be extremely useful for cancer immunotherapy studies, allowing visualization of immune cell/cancer cell interactions, quantifying cytotoxic responses of the modified immune cells, and assessing specificity of on target killing. In conclusion, developing adult immune compromised zebrafish models has provided a much needed step in the evolution of transplantation biology. Engraftment studies using adult immune compromised zebrafish will surely provide unique insights into cancer biology, stem cell and regenerative medicine, and drug discovery, especially in the context of performing large-scale drug studies using oral gavage and visualizing cell responses at single cell resolution in vivo. Dr. Langenau has a patent pending on the immune-compromised zebrafish models. Other authors declare no conflicts of interest. This work is supported by NIH grant R24OD016761, R01CA154923, R01CA215118, R01CA211734, the Liddy Shriver Sarcoma Initiative, the MGH Research Scholars Program, the Millett-O'Neill Sommelier Foundation, the Tosteson & Fund for Medical Discovery Fellowship from MGH (Y.C.), and the Alex's Lemonade Stand Foundation Young Investigator Award (Y.C.).

We report on the characterization of the native form of an American lobster, Homarus americanus, β-defensin-like putative antimicrobial peptide, H. americanus defensin 1 (Hoa-D1), sequenced employing top-down and bottom-up peptidomic strategies using a sensitive, chip-based nanoLC-QTOF-MS/MS instrument. The sequence of Hoa-D1 was determined by mass spectrometry; it was found to contain three disulfide bonds and an amidated C-terminus. The sequence was further validated by searching publicly-accessible H. americanus expressed sequence tag (EST) and transcriptome shotgun assembly (TSA) datasets. Hoa-D1, SYVRScSSNGGDcVYRcYGNIINGAcSGSRVccRSGGGYamide (with c representing a cysteine participating in a disulfide bond), was shown to be related to β-defensin-like peptides previously reported from Panulirus japonicas and Panulirus argus. We found Hoa-D1 in H. americanus hemolymph, hemocytes, the supraoesophageal ganglion (brain), eyestalk ganglia, and pericardial organ extracts, as well as in the plasma of some hemolymph samples. Using discontinuous density gradient separations, we fractionatated hemocytes and localized Hoa-D1 to hemocyte sub-populations. While Hoa-D1 was detected in semigranulocytes and granulocytes using conventional proteomic strategies for analysis, the direct analysis of cell lysates exposed evidence of Hoa-D1 processing, including truncation of the C-terminal tyrosine residue, in the granulocytes, but not semigranulocytes. These measurements demonstrate the insights regarding post-translational modifications and peptide processing that can be revealed through the MS analysis of intact peptides. The identification of Hoa-D1 as a widely-distributed peptide in the lobster suggests the possibility that it may be pleiotropic, with functions in addition to its proposed role as an antimicrobial molecule in the innate immune system.

Branched chain amino acids (BCAA) are comprised of three types of amino acids (valine, leucine, and isoleucine) and are a common type of supplement or a key ingredient in various types of protein supplements. Given their popularity, a laboratory experiment was devised to measure the level of BCAA in various types of milks or supplements. To make this experiment accessible to many, an enzyme assay was adapted to measure the level of BCAAs. It is expected that this project can be done in advanced high school Biology laboratories or college Biochemistry laboratories. The samples are hydrolyzed to break all bonds releasing the free amino acids and the BCAAs are measured using an enzyme assay that detects only these amino acids. In order to validate this method, the samples were also measured for BCAA using reverse phase‐high performance liquid chromatography (RP‐HPLC). It was demonstrated that all three BCAA survived the hydrolysis process with high recoveries. When these amino acids were added to samples, the recoveries were also very high. The results from RP‐HPLC confirm the results from the enzyme assay and demonstrate that this method makes for a viable, accurate and easily accessible laboratory experiment.