Protonophore treatment augments energy expenditure in mice housed at thermoneutrality.

Background: Sub-thermoneutral housing increases facultative thermogenesis in mice, which may mask the pre-clinical efficacy of anti-obesity strategies that target energy expenditure (EE). Here, we quantified the impact of protonophore treatment on whole-body energetics in mice housed at 30°C. Methods: C57BL/6J mice (n = 48, 24M/24F) were housed at 24°C for 2 weeks; 32 (16M/16F) were then transitioned to 30°C for a further 4 weeks. Following 2 weeks acclimation at 30°C, mice (n = 16 per group, 8M/8F) received either normal (0 mg/L; Control) or supplemented (400 mg/L; 2,4-Dinitrophenol [DNP]) drinking water. Mice were singly housed in metabolic cages to determine total EE (TEE) and its components via respiratory gas exchange. Mitochondrial respiratory function of permeabilized liver tissue was determined by high-resolution respirometry. Results: Transitioning mice from 24°C to 30°C reduced TEE and basal EE (BEE) by 16% and 41%, respectively (both P < 0.001). Compared to 30°C controls, TEE was 2.6 kcal/day greater in DNP-treated mice (95% CI: 1.6-3.6 kcal/day, P < 0.001), which was partly due to a 1.2 kcal/day higher BEE in DNP-treated mice (95% CI: 0.6-1.7 kcal/day, P < 0.001). The absolute TEE of 30°C DNP-treated mice was lower than that of mice housed at 24°C in the absence of DNP (DNP: 9.4 ± 0.7 kcal/day vs. 24°C control: 10.4 ± 1.5 kcal/day). DNP treatment reduced overall body fat of females by 2.9 percentage points versus sex-matched controls (95% CI: 1.3%-4.5%, P < 0.001), which was at least partly due to a reduction in inguinal white fat mass. Conclusion: Protonophore treatment markedly increases EE in mice housed at 30°C. The magnitude of change in TEE of mice receiving protonophore treatment at 30°C was smaller than that brought about by transitioning mice from 24°C to 30°C, emphasizing that housing temperature must be considered when assessing anti-obesity strategies that target EE in mice.

Dysregulation of RAS proteostasis by autosomal-dominant LZTR1 mutation induces Noonan syndrome-like phenotypes in mice.

Leucine-zipper-like post translational regulator 1 (LZTR1) is a member of the BTB-Kelch superfamily, which regulates the RAS proteostasis. Autosomal dominant (AD) mutations in LZTR1 have been identified in patients with Noonan syndrome (NS), a congenital anomaly syndrome. However, it remains unclear whether LZTR1 AD mutations regulate the proteostasis of the RAS subfamily molecules or cause NS-like phenotypes in vivo. To elucidate the pathogenesis of LZTR1 mutations, we generated two novel LZTR1 mutation knock-in mice (Lztr1G245R/+ and Lztr1R409C/+), which correspond to the human p.G248R and p.R412C mutations, respectively. LZTR1-mutant male mice exhibit low birth weight, distinctive facial features, and cardiac hypertrophy. Cardiomyocyte size and the expression of RAS subfamily members, including MRAS and RIT1, were significantly increased in the left ventricles (LVs) of mutant male mice. LZTR1 AD mutants did not interact with RIT1 and functioned as dominant-negative forms of wild-type LZTR1. Multi-omics analysis revealed that the MAPK signaling pathway was activated in the LVs of mutant mice. Treatment with the MEK inhibitor trametinib ameliorated cardiac hypertrophy in mutant male mice. These results suggest that MEK/ERK pathway is a therapeutic target for NS-like phenotype resulting from dysfunction of RAS proteostasis by LZTR1 AD mutations.

Osteoclast indices in osteogenesis imperfecta: systematic review and meta-analysis.

Osteogenesis imperfecta (OI) is a rare bone fragility disorder caused by mutations in genes encoding collagen type I or that affect its processing. Alterations in osteoclasts were suggested to contribute to OI pathophysiology. We aimed to systematically identify studies reporting measures of osteoclast formation and function in patients and mouse models of OI, to quantify OI-induced changes. The systematic search of Medline, Ovid, and Web of Science identified 798 unique studies. After screening, we included 23 studies for meta-analysis, reporting osteoclast parameters in 310 patients with OI of 9 different types and 16 studies reporting osteoclast parameters in 406 animals of 11 different OI mouse models. The standardized mean difference with 95% confidence interval (CI) was used as the effect size, and random-effects meta-analysis was performed. In patients with OI, collagen degradation markers were significantly higher compared with age-matched controls, with an effect size of 1.23 (CI: 0.36, 2.10]. Collagen degradation markers were the most elevated in the 3- to 7-year-old age group and in patients with more severe forms of OI. Bone histomorphometry demonstrated the trends for higher osteoclast numbers (1.16; CI: -0.22, 2.55) and osteoclast surface (0.43; CI: -0.63, 1.49), and significantly higher eroded surface (3.24; CI: 0.51, 5.96) compared with age-matched controls. In OI mice, meta-analysis demonstrated significant increases in collagen degradation markers (1.59; CI: 1.07, 2.11), in osteoclast numbers (0.94; CI: 0.50, 1.39), osteoclast surface (0.73; CI: 0.22, 1.23), and eroded surface (1.31; CI: 0.54, 2.08). The largest differences were in OI mice with the mutations in Col1a1 and Col1a2 genes. There were no differences between males and females in clinical or animal studies. Quantitative estimates of changes in osteoclast indices and their variance for patients with OI are important for planning future studies. We confirmed that similar changes are observed in mice with OI, supporting their translational utility.

Toward a CRISPR-based mouse model of Vhl-deficient clear cell kidney cancer: Initial experience and lessons learned.

CRISPR is revolutionizing the ability to do somatic gene editing in mice for the purpose of creating new cancer models. Inactivation of the VHL tumor suppressor gene is the signature initiating event in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). Such tumors are usually driven by the excessive HIF2 activity that arises when the VHL gene product, pVHL, is defective. Given the pressing need for a robust immunocompetent mouse model of human ccRCC, we directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL and other known/suspected ccRCC tumor suppressor genes into the kidneys of C57BL/6 mice under conditions where Cas9 was under the control of one of two different kidney-specific promoters (Cdh16 or Pax8) to induce kidney tumors. An AAV targeting Vhl, Pbrm1, Keap1, and Tsc1 reproducibly caused macroscopic ccRCCs that partially resembled human ccRCC tumors with respect to transcriptome and cell of origin and responded to a ccRCC standard-of-care agent, axitinib. Unfortunately, these tumors, like those produced by earlier genetically engineered mouse ccRCCs, are HIF2 independent.

ERK signaling promotes resistance to TRK kinase inhibition in NTRK fusion-driven glioma mouse models.

Pediatric-type high-grade gliomas frequently harbor gene fusions involving receptor tyrosine kinase genes, including neurotrophic tyrosine kinase receptor (NTRK) fusions. Clinically, these tumors show high initial response rates to tyrosine kinase inhibition but ultimately recur due to the accumulation of additional resistance-conferring mutations. Here, we develop a series of genetically engineered mouse models of treatment-naive and -experienced NTRK1/2/3 fusion-driven gliomas. All tested NTRK fusions are oncogenic in vivo. The NTRK variant, N-terminal fusion partners, and resistance-associated point mutations all influence tumor histology and aggressiveness. Additional tumor suppressor losses greatly enhance tumor aggressiveness. Treatment with TRK kinase inhibitors significantly extends the survival of NTRK fusion-driven glioma mice, but fails to fully eradicate tumors, leading to recurrence upon treatment discontinuation. Finally, we show that ERK activation promotes resistance to TRK kinase inhibition and identify MEK inhibition as a potential combination therapy. These models will be invaluable tools to study therapy resistance of NTRK fusion tumors.

Mouse Cardiovascular Imaging.

The mouse is the mammalian model of choice for investigating cardiovascular biology, given our ability to manipulate it by genetic, pharmacologic, mechanical, and environmental means. Imaging is an important approach to phenotyping both function and structure of cardiac and vascular components. This review details commonly used imaging approaches, with a focus on echocardiography and magnetic resonance imaging, with brief overviews of other imaging modalities. In this update, we also emphasize the importance of rigor and reproducibility in imaging approaches, experimental design, and documentation. Finally, we briefly outline emerging imaging approaches but caution that reliability and validity data may be lacking. © 2024 Wiley Periodicals LLC.

Arrested development: the dysfunctional life history of medulloblastoma.

Medulloblastoma is a heterogeneous embryonal tumor of the cerebellum comprised of four distinct molecular subgroups that differ in their developmental origins, genomic landscapes, clinical presentation, and survival. Recent characterization of the human fetal cerebellum at single-cell resolution has propelled unprecedented insights into the cellular origins of medulloblastoma subgroups, including those underlying previously elusive groups 3 and 4. In this review, the molecular pathogenesis of medulloblastoma is examined through the lens of cerebellar development. In addition, we discuss how enhanced understanding of medulloblastoma origins has the potential to refine disease modeling for the advancement of treatment and outcomes.

The Integral Role of Fibronectin in Skeletal Morphogenesis and Pathogenesis.

Fibronectin (FN) serves as a critical organizer of extracellular matrix networks in two principal isoforms, the plasma FN and the cellular FN. While FN's pivotal role in various organ systems, including the blood vasculature, is well-established, its contribution to the development of the skeletal system is much less explored. Furthermore, the pathomechanisms of spondyloepiphyseal dysplasia caused by FN mutations remain elusive. In this minireview, we discuss findings from our recent two studies using i) an iPSC-based cell culture model to explore how FN mutations in spondyloepiphyseal dysplasia impact mesenchymal cell differentiation into chondrocytes and ii) conditional FN knockout mouse models to determine the physiological roles of FN isoforms during postnatal skeletal development. The data revealed that FN mutations cause severe intracellular and matrix defects in mesenchymal cells and impair their ability to differentiate into chondrocytes. The findings further demonstrate the important roles of both FN isoforms in orchestrating regulated chondrogenesis during skeletal development. We critically discuss the findings in the context of the existing literature.

Compensatory changes after spinal cord injury in a remyelination deficient mouse model.

The development of therapeutic strategies to reduce impairments following spinal cord injury (SCI) motivates an active area of research, because there are no effective therapies. One strategy is to address injury-induced demyelination of spared axons by promoting endogenous or exogenous remyelination. However, previously, we showed that new myelin was not necessary to regain hindlimb stepping following moderate thoracic spinal cord contusion in 3-month-old mice. The present analysis investigated two potential mechanisms by which animals can re-establish locomotion in the absence of remyelination: compensation through intact white matter and conduction through spared axons. We induced a severe contusion injury to reduce the spared white matter rim in the remyelination deficient model, with no differences in recovery between remyelination deficient animals and injured littermate controls. We investigated the nodal properties of the axons at the lesion and found that in the remyelination deficient model, axons express the Nav1.2 voltage-gated sodium channel, a sub-type not typically expressed at mature nodes of Ranvier. In a moderate contusion injury, conduction velocities through the lesions of remyelination deficient animals were similar to those in animals with the capacity to remyelinate after injury. Detailed gait analysis and kinematics reveal subtle differences between remyelination deficient animals and remyelination competent controls, but no worse deficits. It is possible that upregulation of Nav1.2 channels may contribute to establishing conduction through the lesion. This conduction could contribute to compensation and regained motor function in mouse models of SCI. Such compensatory mechanism may have implications for interpreting efficacy results for remyelinating interventions in mice and the development of therapies for improving recovery following SCI.

Experimental models for preclinical research in kidney disease.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are significant public health issues associated with a long-term increase in mortality risk, resulting from various etiologies including renal ischemia, sepsis, drug toxicity, and diabetes mellitus. Numerous preclinical models have been developed to deepen our understanding of the pathophysiological mechanisms and therapeutic approaches for kidney diseases. Among these, rodent models have proven to be powerful tools in the discovery of novel therapeutics, while the development of kidney organoids has emerged as a promising advancement in the field. This review provides a comprehensive analysis of the construction methodologies, underlying biological mechanisms, and recent therapeutic developments across different AKI and CKD models. Additionally, this review summarizes the advantages, limitations, and challenges inherent in these preclinical models, thereby contributing robust evidence to support the development of effective therapeutic strategies.

HSF1 is a prognostic determinant and therapeutic target in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a lethal primary liver tumor characterized by clinical aggressiveness, poor prognosis, and scarce therapeutic possibilities. Therefore, new treatments are urgently needed to render this disease curable. Since cumulating evidence supports the oncogenic properties of the Heat Shock Factor 1 (HSF1) transcription factor in various cancer types, we investigated its pathogenetic and therapeutic relevance in iCCA. METHODS: Levels of HSF1 were evaluated in a vast collection of iCCA specimens. The effects of HSF1 inactivation on iCCA development in vivo were investigated using three established oncogene-driven iCCA mouse models. In addition, the impact of HSF1 suppression on tumor cells and tumor stroma was assessed in iCCA cell lines, human iCCA cancer-associated fibroblasts (hCAFs), and patient-derived organoids. RESULTS: Human preinvasive, invasive, and metastatic iCCAs displayed widespread HSF1 upregulation, which was associated with a dismal prognosis of the patients. In addition, hydrodynamic injection of a dominant-negative form of HSF1 (HSF1dn), which suppresses HSF1 activity, significantly delayed cholangiocarcinogenesis in AKT/NICD, AKT/YAP, and AKT/TAZ mice. In iCCA cell lines, iCCA hCAFs, and patient-derived organoids, administration of the HSF1 inhibitor KRIBB-11 significantly reduced proliferation and induced apoptosis. Cell death was profoundly augmented by concomitant administration of the Bcl-xL/Bcl2/Bcl-w inhibitor ABT-263. Furthermore, KRIBB-11 reduced mitochondrial bioenergetics and glycolysis of iCCA cells. CONCLUSIONS: The present data underscore the critical pathogenetic, prognostic, and therapeutic role of HSF1 in cholangiocarcinogenesis.

Cognitive Effects of Simulated Galactic Cosmic Radiation Are Mediated by ApoE Status, Sex, and Environment in APP Knock-In Mice.

Cosmic radiation experienced during space travel may increase the risk of cognitive impairment. While simulated galactic cosmic radiation (GCRsim) has led to memory deficits in wildtype (WT) mice, it has not been investigated whether GCRsim in combination with genetic risk factors for Alzheimer's disease (AD) worsens memory further in aging mice. Here, we investigated the central nervous system (CNS) effects of 0 Gy (sham) or 0.75 Gy five-ion GCRsim or 2 Gy gamma radiation (IRR) in 14-month-old female and male APPNL-F/NL-F knock-in (KI) mice bearing humanized ApoE3 or ApoE4 (APP;E3F and APP;E4F). As travel to a specialized facility was required for irradiation, both traveled sham-irradiated C57BL/6J WT and KI mice and non-traveled (NT) KI mice acted as controls for potential effects of travel. Mice underwent four behavioral tests at 20 months of age and were euthanized for pathological and biochemical analyses 1 month later. Fecal samples were collected pre- and post-irradiation at four different time points. GCRsim seemed to impair memory in male APP;E3F mice compared to their sham counterparts. Travel tended to improve cognition in male APP;E3F mice and lowered total Aß in female and male APP;E3F mice compared to their non-traveled counterparts. Sham-irradiated male APP;E4F mice accumulated more fibrillar amyloid than their APP;E3F counterparts. Radiation exposure had only modest effects on behavior and brain changes, but travel-, sex-, and genotype-specific effects were seen. Irradiated mice had immediate and long-term differences in their gut bacterial composition that correlated to Alzheimer's disease phenotypes.

Prion protein pathology in Ubiquilin 2 models of ALS.

Mutations in UBQLN2 cause ALS and frontotemporal dementia (FTD). The pathological signature in UBQLN2 cases is deposition of highly unusual types of inclusions in the brain and spinal cord that stain positive for UBQLN2. However, what role these inclusions play in pathogenesis remains unclear. Here we show cellular prion protein (PrPC) is found in UBQLN2 inclusions in both mouse and human neuronal induced pluripotent (IPSC) models of UBQLN2 mutations, evidenced by the presence of aggregated forms of PrPC with UBQLN2 inclusions. Turnover studies indicated that the P497H UBQLN2 mutation slows PrPC protein degradation and leads to mislocalization of PrPC in the cytoplasm. Immunoprecipitation studies indicated UBQLN2 and PrPC bind together in a complex. The abnormalities in PrPC caused by UBQLN2 mutations may be relevant in disease pathogenesis.

Pericyte phenotype switching alleviates immunosuppression and sensitizes vascularized tumors to immunotherapy in preclinical models.

T cell-based immunotherapies are a promising therapeutic approach for multiple malignancies, but their efficacy is limited by tumor hypoxia arising from dysfunctional blood vessels. Here, we report that cell-intrinsic properties of a single vascular component, namely the pericyte, contribute to the control of tumor oxygenation, macrophage polarization, vessel inflammation, and T cell infiltration. Switching pericyte phenotype from a synthetic to a differentiated state reverses immune suppression and sensitizes tumors to adoptive T cell therapy, leading to regression of melanoma in mice. In melanoma patients, improved survival is correlated with enhanced pericyte maturity. Importantly, pericyte plasticity is regulated by signaling pathways converging on Rho kinase activity, with pericyte maturity being inducible by selective low-dose therapeutics that suppress pericyte MEK, AKT, or notch signaling. We also show that low-dose targeted anticancer therapy can durably change the tumor microenvironment without inducing adaptive resistance, creating a highly translatable pathway for redosing anticancer targeted therapies in combination with immunotherapy to improve outcome.

Cellular and Immunological Analysis of 2D2/Th Hybrid Mice Prone to Experimental Autoimmune Encephalomyelitis in Comparison with 2D2 and Th Lines.

Previously, we described the mechanisms of development of autoimmune encephalomyelitis (EAE) in 3-month-old C57BL/6, Th, and 2D2 mice. The faster and more profound spontaneous development of EAE with the achievement of deeper pathology occurs in hybrid 2D2/Th mice. Here, the cellular and immunological analysis of EAE development in 2D2/Th mice was carried out. In Th, 2D2, and 2D2/Th mice, the development of EAE is associated with a change in the differentiation profile of hemopoietic bone marrow stem cells, which, in 2D2/Th, differs significantly from 2D2 and Th mice. Hybrid 2D2/Th mice demonstrate a significant difference in these changes in all strains of mice, leading to the production of antibodies with catalytic activities, known as abzymes, against self-antigens: myelin oligodendrocyte glycoprotein (MOG), DNA, myelin basic protein (MBP), and five histones (H1-H4) hydrolyze these antigens. There is also the proliferation of B and T lymphocytes in different organs (blood, bone marrow, thymus, spleen, lymph nodes). The patterns of changes in the concentration of antibodies and the relative activity of abzymes during the spontaneous development of EAE in the hydrolysis of these immunogens are significantly or radically different for the three lines of mice: Th, 2D2, and 2D2/Th. Several factors may play an essential role in the acceleration of EAE in 2D2/Th mice. The treatment of mice with MOG accelerates the development of EAE pathology. In the initial period of EAE development, the concentration of anti-MOG antibodies in 2D2/Th is significantly higher than in Th (29.1-fold) and 2D2 (11.7-fold). As shown earlier, antibodies with DNase activity penetrate cellular and nuclear membranes and activate cell apoptosis, stimulating autoimmune processes. In the initial period of EAE development, the concentration of anti-DNA antibodies in 2D2/Th hybrids is higher than in Th (4.6-fold) and 2D2 (25.7-fold); only 2D2/Th mice exhibited a very strong 10.6-fold increase in the DNase activity of IgGs during the development of EAE. Free histones in the blood are cytotoxic and stimulate the development of autoimmune diseases. Only in 2D2/Th mice, during different periods of EAE development, was a sharp increase in the anti-antibody activity in the hydrolysis of some histones observed.

TERT upregulation promotes cell proliferation via degradation of p21 and increases carcinogenic potential.

Telomerase reverse transcriptase (TERT) gene aberration is detectable in >80% of cases with hepatocellular carcinoma (HCC). TERT reactivation is essential for cellular immortalization because it stabilizes telomere length, although the role of TERT in hepatocarcinogenesis remains unelucidated. To elucidate the significance of aberrant TERT expression in hepatocytes in inflammation-associated hepatocarcinogenesis, we generated Alb-Cre;TertTg mice, which overexpress TERT in the liver and examined their phenotype during chronic inflammation. Based on transcriptome data from the liver tissue of Alb-Cre;TertTg mice, we examined the role of TERT in hepatocarcinogenesis in vitro. We also evaluated the relationship between TERT and cell-cycle-related molecules, including p21, in HCC samples. The liver tumor development rate was increased by TERT overexpression during chronic inflammation, especially in the absence of p53 function. Gene set enrichment analysis of liver tissues revealed that gene sets related to TNF-NFκB signaling, cell cycle, and apoptosis were upregulated in Alb-Cre;TertTg liver. A luciferase reporter assay and immunoprecipitation revealed that TERT interacted with NFκB p65 and enhanced NFκB promoter activity. On the other hand, TERT formed protein complexes with p21, cyclin A2, and cyclin E and promoted ubiquitin-mediated degradation of p21, specifically in the G1 phase. In the clinical HCC samples, TERT was highly expressed but p21 was conversely downregulated, and TERT expression was associated with the upregulation of molecules related to the cell cycle. Taken together, the aberrant upregulation of TERT increased NFκB promoter activity and promoted cell cycle progression via p21 ubiquitination, leading to hepatocarcinogenesis. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Loss of p53 and SMAD4 induces adenosquamous subtype pancreatic cancer in the absence of an oncogenic KRAS mutation.

Pancreatic cancer is associated with an oncogenic KRAS mutation in approximately 90% of cases. However, a non-negligible proportion of pancreatic cancer cases harbor wild-type KRAS (KRAS-WT). This study establishes genetically engineered mouse models that develop spontaneous pancreatic cancer in the context of KRAS-WT. The Trp53loxP/loxP;Smad4loxP/loxP;Pdx1-Cre (PPSSC) mouse model harbors KRAS-WT and loss of Trp53/Smad4. The Trp53loxP/loxP;Tgfbr2loxP/loxP;Pdx1-Cre (PPTTC) mouse model harbors KRAS-WT and loss of Trp53/Tgfbr2. We identify that either Trp53/Smad4 loss or Trp53/Tgfbr2 loss can induce spontaneous pancreatic tumor formation in the absence of an oncogenic KRAS mutation. The Trp53/Smad4 loss and Trp53/Tgfbr2 loss mouse models exhibit distinct pancreatic tumor histological features, as compared to oncogenic KRAS-driven mouse models. Furthermore, KRAS-WT pancreatic tumors with Trp53/Smad4 loss reveal unique histological features of pancreatic adenosquamous carcinoma (PASC). Single-cell RNA sequencing (scRNA-seq) analysis reveals the distinct tumor immune microenvironment landscape of KRAS-WT (PPSSC) pancreatic tumors as compared with that of oncogenic KRAS-driven pancreatic tumors.

Ultra-high-resolution mapping of myelin and g-ratio in a panel of Mbp enhancer-edited mouse strains using microstructural MRI.

Non-invasive myelin water fraction (MWF) and g-ratio mapping using microstructural MRI have the potential to offer critical insights into brain microstructure and our understanding of neuroplasticity and neuroinflammation. By leveraging a unique panel of variably hypomyelinating mouse strains, we validated a high-resolution, model-free image reconstruction method for whole-brain MWF mapping. Further, by employing a bipolar gradient echo MRI sequence, we achieved high spatial resolution and robust mapping of MWF and g-ratio across the whole mouse brain. Our regional white matter-tract specific analyses demonstrated a graded decrease in MWF in white matter tracts which correlated strongly with myelin basic protein gene (Mbp) mRNA levels. Using these measures, we derived the first sensitive calibrations between MWF and Mbp mRNA in the mouse. Minimal changes in axonal density supported our hypothesis that observed MWF alterations stem from hypomyelination. Overall, our work strongly emphasizes the potential of non-invasive, MRI-derived MWF and g-ratio modeling for both preclinical model validation and ultimately translation to humans.

The Role of Microsurgery and Fluorescent-reporter Genes in Establishing Mouse Models for Real-Time Imaging of Metastatic Cancer-Cell Trafficking and Colony Formation: A Revolutionary and Disruptive Technology for Metastasis Research.

The field of experimental microsurgery was pioneered by the great microsurgeon Sun Lee, who developed the foundation of transplant surgery in the clinic. Dr Lee also played a seminal role in introducing microsurgery to establish mouse models of cancer. In 1990, at the age of 70, Dr Lee demonstrated microsurgery techniques to the mouse-model team at AntiCancer Inc., leading to the development of the surgical orthotopic implant (SOI) technique and the first orthotopic mouse models of cancer that metastasized in a pattern similar to clinical cancer. At the beginning of the present century, one of us (NY) from Kanazawa University School of Medicine became a visiting scientist at AntiCancer to learn SOI and develop mouse models of cancer using cancer cells expressing fluorescent reporter genes, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), in order to image metastatic cancer cells trafficking in real time. Since then, a total of eight young surgeons from Kanazawa University have been visiting researchers at AntiCancer, developing SOI mouse models of cancer to visualize cancer cells in vivo, tracking all stages of metastasis in real time. The present perspective review summarizes this seminal work, which has revolutionized the field of metastasis research.