Mitochondrial diabetes in mice expressing a dominant-negative allele of nuclear respiratory factor-1 (Nrf1) in pancreatic ß-cells.

Genetic polymorphisms in nuclear respiratory factor-1 (Nrf1), a key transcriptional regulator of nuclear-encoded mitochondrial proteins, have been linked to diabetes. Homozygous deletion of Nrf1 is embryonic lethal in mice. Our goal was to generate mice with ß-cell-specific reduction in NRF1 function to investigate the relationship between NRF1 and diabetes. We report the generation of mice expressing a dominant-negative allele of Nrf1 (DNNRF1) in pancreatic ß-cells. Heterozygous transgenic mice had high fed blood glucose levels detected at 3 wks of age, which persisted through adulthood. Plasma insulin levels in DNNRF1 transgenic mice were reduced, while insulin sensitivity remained intact in young animals. Islet size was reduced with increased numbers of apoptotic cells, and insulin content in islets by immunohistochemistry was low. Glucose-stimulated insulin secretion in isolated islets was reduced in DNNRF1-mice, but partially rescued by KCl, suggesting that decreased mitochondrial function contributed to the insulin secretory defect. Electron micrographs demonstrated abnormal mitochondrial morphology in ß-cells. Expression of NRF1 target genes Tfam, Tfb1m and Tfb2m, and islet cytochrome c oxidase and succinate dehydrogenase activities were reduced in DNNRF1-mice. Rescue of mitochondrial function with low level activation of transgenic c-Myc in ß-cells was sufficient to restore ß-cell mass and prevent diabetes. This study demonstrates that reduced NRF1 function can lead to loss of ß-cell function and establishes a model to study the interplay between regulators of bi-genomic gene transcription in diabetes.

Letters or not, here we come! A communal perspective on credentials needed for a productive career in veterinary pathology.

Veterinary pathology credentials serve as a concise means attesting to educational attainments and experiences indicating a readiness for professional practice. Given the cost, time, and stress associated with obtaining different qualifications, pathologists must consider what credentials enhance their readiness. In this commentary, the authors describe how their various degrees and certifications have facilitated their individual and organizational success. The minimum credentials for proficient veterinary pathology practice are a veterinary medical degree (DVM or equivalent) and advanced pathology training (residency and/or on-the-job "apprenticeship") ideally culminating in board certification in pathology (American College of Veterinary Pathologists [ACVP] diplomate status or equivalent). Graduate degrees (MS, PhD, MPH, etc) and/or other qualifications in allied biomedical fields (eg, board certification in internal medicine, laboratory animal medicine, poultry medicine, preventive medicine, or toxicology) may improve employability by affirming specialty knowledge in another complementary discipline. The authors note that pathology positions may be obtained without a long list of degrees or certifications, and that more credentials may provide occupational flexibility for some employers. However, a good work ethic, experience in the field, ability to adapt to changes, job satisfaction, good attitude, and demonstrated productivity are also important, and indeed, they are often the paramount criteria for career success as a veterinary pathologist.

211At-Labeled Anti-CD45 Antibody as a Nonmyeloablative Conditioning for Canine DLA-Haploidentical Stem Cell Transplantation.

The α-emitter 211At deposits a high amount of energy within a few cell diameters, resulting in irreparable DNA double-strand breaks while minimizing off-target toxicity. We investigated the use of the 211At-labeled anti-CD45 monoclonal antibody (mAb) 211At-CD45-B10 as a nonmyeloablative conditioning regimen for dog-leukocyte-antigen-haploidentical hematopoietic cell transplantation. Methods: Seventeen healthy dogs were injected with either a 0.50 (n = 14) or 0.75 (n = 3) mg/kg dose of anti-CD45 mAb labeled with 211At (8.436-23.199 MBq [0.228-0.627 mCi/kg]) on day -3. Peripheral blood stem cells from dog-leukocyte-antigen-haploidentical donors were given on day 0. Peripheral blood chimerism was calculated by polymerase chain reaction assays, and blood clearance of the radioimmunoconjugate was studied using enzyme-linked immunosorbent assay and radioactivity measurements of serial blood samples. Results: All dogs achieved donor chimerism by day 28 (range, 27%-100%). The hematopoietic engraftment rate was 100%, though engraftment durability was variable. No difference in absorbed dose to blood was seen for the 2 mAb dosing levels studied. Neutropenia (0-29 cells/µL), lymphocytopenia (36-130 cells/µL), and thrombocytopenia (1.5-9 × 103/µL) with prompt recovery were observed. The main adverse nonhematologic event related to 211At-CD45-B10 was mild reversible transaminitis. Graft-versus-host disease was not seen. Twelve of the 17 dogs survived over 30 d, with donor chimerism ranging from 3% to 99%. Conclusion: The results suggest that nonmyeloablative conditioning with 211At-CD45-B10 could be used in haploidentical hematopoietic cell transplantation though with variable engraftment.

Bioorthogonal non-canonical amino acid tagging to track transplanted human induced pluripotent stem cell-specific proteome.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo. METHODS: In this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts. RESULTS: We demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs. CONCLUSION: The novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.

Physical Activity Decreases Inflammation and Delays the Development of Obesity-Associated Pancreatic Ductal Adenocarcinoma.

Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), a deadly disease with limited preventive strategies. Lifestyle interventions to decrease obesity represent a potential approach to prevent obesity-associated PDAC. In this study, we examined whether decreasing obesity through physical activity (PA) and/or dietary changes could decrease inflammation in humans and prevent obesity-associated PDAC in mice. Comparison of circulating inflammatory-associated cytokines in subjects (overweight and obese) before and after a PA intervention revealed PA lowered systemic inflammatory cytokines. Mice with pancreatic-specific inducible KrasG12D expression were exposed to PA and/or dietary interventions during and after obesity-associated cancer initiation. In mice with concurrent diet-induced obesity and KrasG12D expression, the PA intervention led to lower weight gain, suppressed systemic inflammation, delayed tumor progression, and decreased proinflammatory signals in the adipose tissue. However, these benefits were not as evident when obesity preceded pancreatic KrasG12D expression. Combining PA with diet-induced weight loss (DI-WL) delayed obesity-associated PDAC progression in the genetically engineered mouse model, but neither PA alone nor combined with DI-WL or chemotherapy prevented PDAC tumor growth in orthotopic PDAC models regardless of obesity status. PA led to the upregulation of Il15ra in adipose tissue. Adipose-specific overexpression of Il15 slowed PDAC growth but only in nonobese mice. Overall, our study suggests that PA alone or combined with DI-WL can reduce inflammation and delay obesity-associated PDAC development or progression. Lifestyle interventions that prevent or manage obesity or therapies that target weight loss-related molecular pathways could prevent progression of PDAC. Significance: Physical activity reduces inflammation and induces changes to adipose-related signaling to suppress pancreatic cancer, supporting the potential of obesity management strategies to reduce the risk of developing pancreatic cancer. See related commentary by Sogunro and Muzumdar, p. 2935.

Mitochondrial diabetes in mice expressing a dominant-negative allele of nuclear respiratory factor-1 ( Nrf1 ) in pancreatic ß-cells.

Genetic polymorphisms in nuclear respiratory factor-1 ( NRF1 ), a key transcriptional regulator of nuclear-encoded mitochondrial proteins, have been linked to diabetes. Homozygous deletion of Nrf1 is embryonic lethal in mice. Our goal was to generate mice with ß-cell-specific reduction in NRF1 function to investigate the relationship between NRF1 and diabetes. We report the generation of mice expressing a dominant-negative allele of Nrf1 (DNNRF1) in pancreatic ß-cells. Heterozygous transgenic mice had high fed blood glucose levels detected at 3 wks of age, which persisted through adulthood. Plasma insulin levels in DNNRF1 transgenic mice were reduced, while insulin sensitivity remained intact in young animals. Islet size was reduced with increased numbers of apoptotic cells, and insulin content in islets by immunohistochemistry was low. Glucose-stimulated insulin secretion in isolated islets was reduced in DNNRF1-mice, but partially rescued by KCl, suggesting that decreased mitochondrial function contributed to the insulin secretory defect. Electron micrographs demonstrated abnormal mitochondrial morphology in ß- cells. Expression of NRF1 target genes Tfam , T@1m and T@2m , and islet cytochrome c oxidase and succinate dehydrogenase activities were reduced in DNNRF1-mice. Rescue of mitochondrial function with low level activation of transgenic c-Myc in ß-cells was sufficient to restore ß-cell mass and prevent diabetes. This study demonstrates that reduced NRF1 function can lead to loss of ß-cell function and establishes a model to study the interplay between regulators of bi- genomic gene transcription in diabetes.

Mammary gland development and EDC-driven cancer susceptibility in mesenchymal ERα-knockout mice.

Development of the mammary gland requires both proper hormone signaling and cross talk between the stroma and epithelium. While estrogen receptor (ERα) expression in the epithelium is essential for normal gland development, the role of this receptor in the stroma is less clear. Moreover, several lines of evidence suggest that mouse phenotypes of in utero exposure to endocrine disruption act through mesenchymal ERα in the developing fetus. We utilized a Twist2-cre mouse line to knock out mesenchymal ERα. Herein, we assessed mammary gland development in the context of mesenchymal ERα deletion. We also tested the effect of in utero bisphenol A (BPA) exposure to alter the tumor susceptibility in the mouse mammary tumor virus-neu (MMTV-neu) breast cancer mouse model. Mesenchymal ERα deletion resulted in altered reproductive tract development and atypical cytology associated with estrous cycling. The mammary gland demonstrated mature epithelial extension unlike complete ERα-knockout mice, but ductal extension was delayed and reduced compared to ERα-competent mice. Using the MMTV-Neu cancer susceptibility model, ERα-intact mice exposed to BPA had reduced tumor-free survival and overall survival compared to BPA-exposed mice having mesenchymal ERα deletion. This difference is specific for BPA exposure as vehicle-treated animals had no difference in tumor development between mice expressing and not expressing mesenchymal ERα. These data demonstrate that mesenchymal ERα expression is not required for ductal extension, nor does it influence cancer risk in this mouse model but does influence the cancer incidence associated with in utero BPA exposure.

Physical Activity Delays Obesity-Associated Pancreatic Ductal Adenocarcinoma in Mice and Decreases Inflammation.

BACKGROUND & AIMS: Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), a deadly disease with limited preventive strategies. Lifestyle interventions to decrease obesity might prevent obesity-associated PDAC. Here, we examined whether decreasing obesity by increased physical activity (PA) and/or dietary changes would decrease inflammation in humans and prevent PDAC in mice. METHODS: Circulating inflammatory-associated cytokines of overweight and obese subjects before and after a PA intervention were compared. PDAC pre-clinical models were exposed to PA and/or dietary interventions after obesity-associated cancer initiation. Body composition, tumor progression, growth, fibrosis, inflammation, and transcriptomic changes in the adipose tissue were evaluated. RESULTS: PA decreased the levels of systemic inflammatory cytokines in overweight and obese subjects. PDAC mice on a diet-induced obesity (DIO) and PA intervention, had delayed weight gain, decreased systemic inflammation, lower grade pancreatic intraepithelial neoplasia lesions, reduced PDAC incidence, and increased anti-inflammatory signals in the adipose tissue compared to controls. PA had additional cancer prevention benefits when combined with a non-obesogenic diet after DIO. However, weight loss through PA alone or combined with a dietary intervention did not prevent tumor growth in an orthotopic PDAC model. Adipose-specific targeting of interleukin (IL)-15, an anti-inflammatory cytokine induced by PA in the adipose tissue, slowed PDAC growth. CONCLUSIONS: PA alone or combined with diet-induced weight loss delayed the progression of PDAC and reduced systemic and adipose inflammatory signals. Therefore, obesity management via dietary interventions and/or PA, or modulating weight loss related pathways could prevent obesity-associated PDAC in high-risk obese individuals.

Supporting diversity, equity, inclusion, and belonging to strengthen and position the veterinary profession for service, sustainability, excellence, and impact.

Advancing equality and equity in society is creating positive change, and the time has come to critically evaluate veterinary medicine, which, by all metrics, lacks diversity. To keep pace with increasingly diverse demographics and recent surges in pet ownership among all racial/ethnic groups, significant efforts to enhance diversity, equity, inclusion, and belonging (DEIB) must occur in veterinary colleges and the profession. Recruiting more underrepresented students, building pipelines for diverse faculty/staff, and creating inclusive, welcoming environments where all can thrive are critical steps toward enhancing DEIB within our organizations and profession. Our goal is to share experiences and lessons learned from our intentional commitment to strengthen DEIB, with the hope that our journey will be helpful to others. Increasing diversity in the veterinary profession will be facilitated through removing barriers, creating inclusive work environments where all people feel they belong, and ensuring fair and equitable hiring and personnel management practices. These steps should in turn improve access and quality of veterinary care, ensure we are more representative of the communities we serve, increase revenue, and preserve the human-animal bond. "You cannot change any society unless you take responsibility for it, unless you see yourself belonging to it, and responsible for changing it." - Grace Lee Boggs.

KMT2D loss drives aggressive tumor phenotypes in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most lethal skin cancer. Due to ultraviolet light-induced damage, cSCCs have a high mutation rate, but some genes are more frequently mutated in aggressive cSCCs. Lysine-specific histone methyltransferase 2D (KMT2D) has a two-fold higher mutation frequency in metastatic cSCCs relative to primary non-metastatic associated cSCCs. The role of KMT2D in more aggressive phenotypes in cSCC is uncharacterized. Studies of other tumor types suggest that KMT2D acts to suppress tumor development. To determine whether KMT2D loss has an impact on tumor characteristics, we disrupted KMT2D in a cSCC cell line using CRISPR-cas9 and performed phenotypic analyses. KMT2D loss modestly increased cell proliferation and colony formation (1.4- and 1.6-fold respectively). Cells lacking KMT2D showed increased rates of migration and faster cell cycle progression. In xenograft models, tumors with KMT2D loss showed slight increases in mitotic indices. Collectively, these findings suggest that KMT2D loss-of-function mutations may promote more aggressive and invasive behaviors in cSCC, suggesting that KMT2D-related pathways could be targets for cancer therapies. Future studies to determine the downstream genes and mechanism of phenotypic effect are needed.

The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer.

BACKGROUND: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. METHODS: The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. RESULTS: Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. CONCLUSIONS: Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.

Facilitative lysosomal transport of bile acids alleviates ER stress in mouse hematopoietic precursors.

Mutations in human equilibrative nucleoside transporter 3 (ENT3) encoded by SLC29A3 results in anemia and erythroid hypoplasia, suggesting that ENT3 may regulate erythropoiesis. Here, we demonstrate that lysosomal ENT3 transport of taurine-conjugated bile acids (TBA) facilitates TBA chemical chaperone function and alleviates endoplasmic reticulum (ER) stress in expanding mouse hematopoietic stem and progenitor cells (HSPCs). Slc29a3-/- HSPCs accumulate less TBA despite elevated levels of TBA in Slc29a3-/- mouse plasma and have elevated basal ER stress, reactive oxygen species (ROS), and radiation-induced apoptosis. Reintroduction of ENT3 allows for increased accumulation of TBA into HSPCs, which results in TBA-mediated alleviation of ER stress and erythroid apoptosis. Transplanting TBA-preconditioned HSPCs expressing ENT3 into Slc29a3-/- mice increase bone marrow repopulation capacity and erythroid pool size and prevent early mortalities. Together, these findings suggest a putative role for a facilitative lysosomal transporter in the bile acid regulation of ER stress in mouse HSPCs which may have implications in erythroid biology, the treatment of anemia observed in ENT3-mutated human genetic disorders, and nucleoside analog drug therapy.

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Nonhelical Helicobacter pylori Mutants Show Altered Gland Colonization and Elicit Less Gastric Pathology than Helical Bacteria during Chronic Infection.

Half of all humans harbor Helicobacter pylori in their stomachs. Helical cell shape is thought to facilitate H. pylori's ability to bore into the protective mucus layer in a corkscrew-like motion, thereby enhancing colonization of the stomach. H. pylori cell shape mutants show impaired colonization of the mouse stomach, highlighting the importance of cell shape in infection. To gain a deeper understanding of how helical cell morphology promotes host colonization by H. pylori, we used three-dimensional confocal microscopy to visualize the clinical isolate PMSS1 and an isogenic straight-rod mutant (Δcsd6) within thick longitudinal mouse stomach sections. We also performed volumetric image analysis to quantify the number of bacteria residing within corpus and antral glands in addition to measuring total CFU. We found that straight rods show attenuation during acute colonization of the stomach (1 day or 1 week postinfection) as measured by total CFU. Our quantitative imaging revealed that wild-type bacteria extensively colonized antral glands at 1 week postinfection, while csd6 mutants showed variable colonization of the antrum at this time point. During chronic infection (1 or 3 months postinfection), total CFU were highly variable but similar for wild-type and straight rods. Both wild-type and straight rods persisted and expanded in corpus glands during chronic infection. However, the straight rods showed reduced inflammation and disease progression. Thus, helical cell shape contributes to tissue interactions that promote inflammation during chronic infection, in addition to facilitating niche acquisition during acute infection.

Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice.

Insertional mutagenesis is a powerful means of identifying cancer drivers in animal models. We used the Sleeping Beauty (SB) transposon/transposase system to identify activated oncogenes in hematologic cancers in wild-type mice and mice that express a stabilized cyclin E protein (termed cyclin ET74AT393A). Cyclin E governs cell division and is misregulated in human cancers. Cyclin ET74AT393A mice develop ineffective erythropoiesis that resembles early-stage human myelodysplastic syndrome, and we sought to identify oncogenes that might cooperate with cyclin E hyperactivity in leukemogenesis. SB activation in hematopoietic precursors caused T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL). Analysis of >12,000 SB integration sites revealed markedly different oncogene activations in EL and T-ALL: Notch1 and Ikaros were most common in T-ALL, whereas ETS transcription factors (Erg and Ets1) were targeted in most ELs. Cyclin E status did not impact leukemogenesis or oncogene activations. Whereas most SB insertions were lost during culture of EL cell lines, Erg insertions were retained, indicating Erg's key role in these neoplasms. Surprisingly, cyclin ET74AT393A conferred growth factor independence and altered Erg-dependent differentiation in EL cell lines. These studies provide new molecular insights into erythroid leukemia and suggest potential therapeutic targets for human leukemia.

Keeping Score: Semiquantitative and Quantitative Scoring Approaches to Genetically Engineered and Xenograft Mouse Models of Cancer.

There is a growing need to quantitate or "score" lesions in mouse models of human disease, for correlation with human disease and to establish their clinical relevance. Several standard semiquantitative scoring schemes have been adapted for nonneoplastic lesions; similarly, the pathologist must carefully select an approach to score mouse models of cancer. Genetically engineered mouse models with a continuum of precancerous and cancerous lesions and xenogeneic models of various derivations present unique challenges for the pathologist. Important considerations include experimental design, understanding of the human disease being modeled, standardized classification of lesions, and approaches for semiquantitative and/or quantitative scoring in the model being evaluated. Quantification should be considered for measuring the extent of neoplasia and expression of tumor biomarkers. Semiquantitative scoring schemes have been devised that include severity, frequency, and distribution of lesions. Although labor-intensive, scoring mouse models of cancer provides numerical data that enable statistical analysis and greater translational impact.

Pathology Principles and Practices for Analysis of Animal Models.

Over 60% of NIH extramural funding involves animal models, and approximately 80% to 90% of these are mouse models of human disease. It is critical to translational research that animal models are accurately characterized and validated as models of human disease. Pathology analysis, including histopathology, is essential to animal model studies by providing morphologic context to in vivo, molecular, and biochemical data; however, there are many considerations when incorporating pathology endpoints into an animal study. Mice, and in particular genetically modified models, present unique considerations because these modifications are affected by background strain genetics, husbandry, and experimental conditions. Comparative pathologists recognize normal pathobiology and unique phenotypes that animals, including genetically modified models, may present. Beyond pathology, comparative pathologists with research experience offer expertise in animal model development, experimental design, optimal specimen collection and handling, data interpretation, and reporting. Critical pathology considerations in the design and use of translational studies involving animals are discussed, with an emphasis on mouse models.

Transposon mutagenesis identifies candidate genes that cooperate with loss of transforming growth factor-beta signaling in mouse intestinal neoplasms.

Colorectal cancer (CRC) results from the accumulation of gene mutations and epigenetic alterations in colon epithelial cells, which promotes CRC formation through deregulating signaling pathways. One of the most commonly deregulated signaling pathways in CRC is the transforming growth factor ß (TGF-ß) pathway. Importantly, the effects of TGF-ß signaling inactivation in CRC are modified by concurrent mutations in the tumor cell, and these concurrent mutations determine the ultimate biological effects of impaired TGF-ß signaling in the tumor. However, many of the mutations that cooperate with the deregulated TGF-ß signaling pathway in CRC remain unknown. Therefore, we sought to identify candidate driver genes that promote the formation of CRC in the setting of TGF-ß signaling inactivation. We performed a forward genetic screen in mice carrying conditionally inactivated alleles of the TGF-ß receptor, type II (Tgfbr2) using Sleeping Beauty (SB) transposon mediated mutagenesis. We used TAPDANCE and Gene-centric statistical methods to identify common insertion sites (CIS) and, thus, candidate tumor suppressor genes and oncogenes within the tumor genome. CIS analysis of multiple neoplasms from these mice identified many candidate Tgfbr2 cooperating genes and the Wnt/ß-catenin, Hippo and MAPK pathways as the most commonly affected pathways. Importantly, the majority of candidate genes were also found to be mutated in human CRC. The SB transposon system provides an unbiased method to identify Tgfbr2 cooperating genes in mouse CRC that are functionally relevant and that may provide further insight into the pathogenesis of human CRC.