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Experimental preclinical models have been a cornerstone of lung cancer translational research. Work in these model systems has provided insights into the biology of lung cancer subtypes and their origins, contributed to our understanding of the mechanisms that underlie tumor progression, and revealed new therapeutic vulnerabilities. Initially patient-derived lung cancer cell lines were the main preclinical models available. The landscape is very different now with numerous preclinical models for research each with unique characteristics. These include genetically engineered mouse models (GEMMs), patient-derived xenografts (PDXs) and three-dimensional culture systems ("organoid" cultures). Here we review the development and applications of these models and describe their contributions to lung cancer research.

Highlights • PDAC cells in the duodenal epithelium mimic intestinal cells and co-opt the basement membrane. • Intramucosal PDAC location is strongly coupled to the classical phenotype and to intestinal traits. • Intratumoral heterogeneity is linked to specific tissue compartments, which shape phenotype plasticity of PDAC cells., Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid tumors. Based on transcriptomic classifiers, basal-like and classical PDAC subtypes have been defined that differ in prognosis. Cells of both subtypes can coexist in individual tumors; however, the contribution of either clonal heterogeneity or microenvironmental cues to subtype heterogeneity is unclear. Here, we report the spatial tumor phenotype dynamics in a cohort of patients in whom PDAC infiltrated the duodenal wall, and identify the duodenal epithelium as a distinct PDAC microniche. Materials and methods We used serial multiplex quantitative immunohistochemistry (smq-IHC) for 24 proteins to phenotypically chart PDAC tumor cells in patients whose tumors infiltrated the duodenal epithelium. Additionally, we used a genetically engineered mouse model to study the PDAC cell phenotype in the small intestinal epithelium in a controlled genetic background. Result We show that pancreatic cancer cells revert to non-destructive growth upon integration into the duodenal epithelium, where they adopt traits of intestinal cell differentiation, associated with phenotypical stabilization of the classical subtype. The integrated tumor cells replace epithelial cells in an adenoma-like manner, as opposed to invasive growth in the submucosa. Finally, we show that this phenomenon is shared between species, by confirming duodenal integration and phenotypic switching in a genetic PDAC mouse model. Discussion Our results identify the duodenal epithelium as a distinct PDAC microniche and tightly link microenvironmental cue to cancer transcriptional subtypes. The phenomenon of “intestinal mimicry” provides a unique opportunity for the systematic investigation of microenvironmental influences on pancreatic cancer plasticity.

Transgenic breast cancer mouse models are critical tools for preclinical studies of human breast cancer. Genetic editing of the murine mammary gland allows for modeling of abnormal genetic events frequently found in human breast cancers. Genetically engineered mouse models (GEMMs) of breast cancer employ tissue-specific genetic manipulation for tumorigenic induction within the mammary tissue. Under the transcriptional control of mammary-specific promoters, transgenic mouse models can simulate spontaneous mammary tumorigenesis by expressing one or more putative oncogenes, such as MYC, HRAS, and PIK3CA. Alternatively, the Cre-Lox system allows for tissue-specific deletion of tumor suppressors, such as p53, Rb1, and Brca1, or specific knock-in of putative oncogenes. Thus, GEMMs can be designed to implement one or more genetic events to induce mammary tumorigenesis. Features of GEMMs, such as age of transgene expression, breeding quality, tumor latency, histopathological characteristics, and propensity for local and distant metastasis, are variable and strain-dependent. This review aims to summarize currently available transgenic breast cancer mouse models that undergo spontaneous mammary tumorigenesis upon genetic manipulation, their varying characteristics, and their individual genetic manipulations that model aberrant signaling events observed in human breast cancers.

Integrin CD103 mediates the adhesion and tissue retention of T cells by binding to E-cadherin which is abundant on epithelial cells. Notably, CD103 is highly expressed on CD8 T cells but conspicuously absent on most CD4 T cells. The mechanism controlling such lineage-specific expression of CD103 remains unclear. Using a series of genetically engineered mouse models, here, we demonstrate that the regulatory mechanism of CD103 expression is distinct between CD4 and CD8 T cells, and that the transcription factor Runx3 plays an important but not an essential role in this process. We further found that the availability of integrin β7 which heterodimerizes with CD103 was necessary but also constrained the surface expression of CD103. Notably, the forced surface expression of CD103 did not significantly alter the thymic development of conventional T cells but severely impaired the generation of MHC-II-restricted TCR transgenic T cells, revealing previously unappreciated aspects of CD103 in the selection and maturation of CD4 T cells. Unlike its effect on CD4 T cell development, however, CD103 overexpression did not significantly affect CD4 T cells in peripheral tissues. Moreover, the frequency and number of CD4 T cells in the small intestine epithelium did not increase even though E-cadherin is highly expressed in this tissue. Collectively, these results suggest that most mature CD4 T cells are refractory to the effects of CD103 expression, and that they presumably utilize CD103-independent pathways to control their tissue retention and residency.

Immunotherapy has enabled remarkable therapeutic responses across cancers of various lineages, albeit with some notable exceptions such as glioblastoma. Several previous misconceptions, which have impaired progress in the past, including the presence and role of the blood-brain barrier and a lack of lymphatic drainage, have been refuted. Nonetheless, a subset of patients with brain metastases but, paradoxically, not the vast majority of those with gliomas are able to respond to immune-checkpoint inhibitors. Immune profiling of samples obtained from patients with central nervous system malignancies using techniques such as mass cytometry and single-cell sequencing along with experimental data from genetically engineered mouse models have revealed fundamental differences in immune composition and immunobiology that not only explain the differences in responsiveness to these agents but also lay the foundations for immunotherapeutic strategies that are applicable to gliomas. Herein, we review the emerging data on the differences in immune cell composition, function and interactions within central nervous system tumours and provide guidance on the development of novel immunotherapies for these historically difficult-to-treat cancers.

Neurofibromatosis Type II (NF2) is an autosomal dominant cancer predisposition syndrome in which germline haploinsufficiency at the NF2 gene confers a greatly increased propensity for tumor development arising from tissues of neural crest derived origin. NF2 encodes the tumor suppressor, Merlin, and its biochemical function is incompletely understood. One well-established function of Merlin is as a negative regulator of group A serine/threonine p21-activated kinases (PAKs). In these studies we explore the role of PAK1 and its closely related paralog, PAK2, both pharmacologically and genetically, in Merlin-deficient Schwann cells and in a genetically engineered mouse model (GEMM) that develops spontaneous vestibular and spinal schwannomas. We demonstrate that PAK1 and PAK2 are both hyper activated in Merlin-deficient murine schwannomas. In preclinical trials, a pan Group A PAK inhibitor, FRAX-1036, transiently reduced PAK1 and PAK2 phosphorylation in vitro, but had insignificant efficacy in vivo. NVS-PAK1-1, a PAK1 selective inhibitor, had a greater but still minimal effect on our GEMM phenotype. However, genetic ablation of Pak1 but not Pak2 reduced tumor formation in our NF2 GEMM. Moreover, germline genetic deletion of Pak1 was well tolerated, while conditional deletion of Pak2 in Schwann cells resulted in significant morbidity and mortality. These data support the further development of PAK1-specific small molecule inhibitors and the therapeutic targeting of PAK1 in vestibular schwannomas and argue against PAK1 and PAK2 existing as functionally redundant protein isoforms in Schwann cells.

Medulloblastoma is the most common malignant cerebellar tumor in children. Recent technological advances in multilayered ’omics data analysis have revealed 4 molecular subgroups of medulloblastoma (Wingless/int, Sonic hedgehog, Group3, and Group4). (Epi)genomic and transcriptomic profiling on human primary medulloblastomas has shown distinct oncogenic drivers and cellular origin(s) across the subgroups. Despite tremendous efforts to identify the molecular signals driving tumorigenesis, few of the identified targets were druggable; therefore, a further understanding of the etiology of tumors is required to establish effective molecular‐targeted therapies. Chromatin regulators are frequently mutated in medulloblastoma, prompting us to investigate epigenetic changes and the accompanying activation of oncogenic signaling during tumorigenesis. For this purpose, we have used germline and non‐germline genetically engineered mice to model human medulloblastoma and to conduct useful, molecularly targeted, preclinical studies. This review discusses the biological implications of chromatin regulator mutations during medulloblastoma pathogenesis, based on recent in vivo animal studies., Genomic analyses of human medulloblastomas have revealed recurrent mutations of many chromatin regulators. Using germline and non‐germline genetically engineered mouse models, the contribution of these mutations to medulloblastoma formation is being investigated as a hot topic.

Summary There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

Lung squamous cell carcinoma (LUSC) represents a major subtype of non-small cell lung cancer with limited treatment options. Previous studies have elucidated the complex genetic landscape of LUSC and revealed multiple altered genes and pathways. However, in stark contrast to lung adenocarcinoma, few targetable driver mutations have been established so far and targeted therapies for LUSC remain unsuccessful. Immunotherapy has revolutionized LUSC treatment and is currently approved as the new standard of care. To gain a better understanding of the LUSC biology, improved modeling systems are urgently needed. Preclinical models, particularly those mimicking human disease with an intact tumor immune microenvironment, are an invaluable tool to study cancer development and evaluate new therapeutic targets. Here, we discuss recent advances in LUSC preclinical models, with a focus on genetically engineered mouse models (GEMMs) and organoids, in the context of evolving precision medicine and immunotherapy.

Genetically engineered mouse (GEM) models are commonly used in biomedical research. Generating GEMs involve complex set of experimental procedures requiring sophisticated equipment and highly skilled technical staff. Because of these reasons, most research institutes set up centralized core facilities where custom GEMs are created for research groups. Researchers, on the other hand, when they begin thinking about generating GEMs for their research, several questions arise in their minds. For example, what type of model(s) would be best useful for my research, how do I design them, what are the latest technologies and tools available for developing my model(s), and finally how to breed GEMs in my research. As there are several considerations and options in mouse designs, and as it is an expensive and time-consuming endeavor, careful planning upfront can ensure the highest chance of success. In this article, we provide brief answers to several frequently asked questions that arise when researchers begin thinking about generating mouse model(s) for their work.

Recent clinical and preclinical advances have highlighted the existence of a previously hypothesized lymphogenous route of metastasis. However, due to a lack of suitable preclinical modeling tools, its contribution to long-term disease outcome and relevance for therapy remain controversial. Here, we established a genetically engineered mouse model (GEMM) fragment–based tumor model uniquely sustaining a functional network of intratumoral lymphatics that facilitates seeding of fatal peripheral metastases. Multiregimen survival studies and correlative patient data identified primary tumor–derived Angiopoietin-2 (Ang2) as a potent therapeutic target to restrict lymphogenous tumor cell dissemination. Mechanistically, tumor-associated lymphatic endothelial cells (EC), in contrast to blood vascular EC, were found to be critically addicted to the Angiopoietin–Tie pathway. Genetic manipulation experiments in combination with single-cell mapping revealed agonistically acting Ang2–Tie2 signaling as key regulator of lymphatic maintenance. Correspondingly, acute presurgical Ang2 neutralization was sufficient to prolong survival by regressing established intratumoral lymphatics, hence identifying a therapeutic regimen that warrants further clinical evaluation. Significance: Exploiting multiple mouse tumor models including a unique GEMM-derived allograft system in combination with preclinical therapy designs closely matching the human situation, this study provides fundamental insight into the biology of tumor-associated lymphatic EC and defines an innovative presurgical therapeutic window of migrastatic Ang2 neutralization to restrict lymphogenous metastasis. This article is highlighted in the In This Issue feature, p. 211

We previously identified ZNF217 as an oncogenic driver of a subset of osteosarcomas using the Sleeping Beauty (SB) transposon system. Here, we followed up by investigating the genetic role of ZNF217 in osteosarcoma initiation and progression through the establishment of a novel genetically engineered mouse model, in vitro assays, orthotopic mouse studies, and paired these findings with preclinical studies using a small-molecule inhibitor. Throughout, we demonstrate that ZNF217 is coupled to numerous facets of osteosarcoma transformation, including proliferation, cell motility, and anchorage independent growth, and ultimately promoting osteosarcoma growth, progression, and metastasis in part through positive modulation of PI3K–AKT survival signaling. Pharmacologic blockade of AKT signaling with nucleoside analogue triciribine in ZNF217+ orthotopically injected osteosarcoma cell lines reduced tumor growth and metastasis. Our data demonstrate that triciribine treatment may be a relevant and efficacious therapeutic strategy for patients with osteosarcoma with ZNF217+ and p-AKT rich tumors. With the recent revitalization of triciribine for clinical studies in other solid cancers, our study provides a rationale for further evaluation preclinically with the purpose of clinical evaluation in patients with incurable, ZNF217+ osteosarcoma.

Various retroviral and lentiviral vectors have been used for up-the-teat intraductal injection to deliver markers, oncogenes, and other genes into mammary epithelial cells in mice. These methods along with the large number of genetically engineered mouse lines have greatly helped us learn normal breast development and tumorigenesis. Rats are also valuable models for studying human breast development and cancer. However, genetically engineered rats are still uncommon, and previous reports of intraductal injection of retroviral vectors into rats appear to be inefficient in generating mammary tumors. Here, we report, and describe the method for, stably introducing marker genes and oncogenes into mammary glands in rats using intraductal injection of commonly used lentiviral vectors. This method can infect mammary epithelial cells efficiently, and the infected cells can initiate tumorigenesis, including estrogen receptor-positive and hormone-dependent tumors, which are the most common subtype of human breast cancer but are yet still difficult to model in mice. This technique provides another tool for studying formation, prevention, and treatment of breast cancer, especially estrogen receptor-positive breast cancer.

Breast cancer is associated with the second highest cancer-associated deaths worldwide. Therefore, understanding the key events that determine breast cancer progression, modulation of the tumor-microenvironment and metastasis, which is the main cause of cancer-associated death, are of great importance. The mammary specific polyomavirus middle T antigen overexpression mouse model (MMTV-PyMT), first published in 1992, is the most commonly used genetically engineered mouse model (GEMM) for cancer research. Mammary lesions arising in MMTV-PyMT mice follow similar molecular and histological progression as human breast tumors, making it an invaluable tool for cancer researchers and instrumental in understanding tumor biology. In this review, we will highlight key studies that demonstrate the utility of PyMT derived GEMMs in understanding the molecular basis of breast cancer progression, metastasis and highlight its use as a pre-clinical tool for therapeutic discovery.

NME1 is a metastasis-suppressor gene (MSG), capable of suppressing metastatic activity in cell lines of melanoma, breast carcinoma and other cancer origins without affecting their growth in culture or as primary tumours. Herein, we selectively ablated the tandemly arranged Nme1 and Nme2 genes to assess their individual impacts on metastatic activity in a mouse model (HGF:p16−/−) of ultraviolet radiation (UVR)-induced melanoma. Metastatic activity was strongly enhanced in both genders of Nme1- and Nme2-null mice, with stronger activity in females across all genotypes. The study ascribes MSG activity to Nme2 for the first time in an in vivo model of spontaneous cancer, as well as a novel metastasis-suppressor function to Nme1 in the specific context of UVR-induced melanoma.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by initial chemosensitivity followed by emergence of chemoresistant disease. To study roles for MYCN amplification in SCLC progression and chemoresistance, we developed a genetically engineered mouse model of MYCN-overexpressing SCLC. In treatment-naïve mice, MYCN overexpression promoted cell cycle progression, suppressed infiltration of cytotoxic T cells, and accelerated SCLC. MYCN overexpression also suppressed response to cisplatin–etoposide chemotherapy, with similar findings made upon MYCL overexpression. We extended these data to genetically perturb chemosensitive patient-derived xenograft (PDX) models of SCLC. In chemosensitive PDX models, overexpression of either MYCN or MYCL also conferred a switch to chemoresistance. To identify therapeutic strategies for MYCN-overexpressing SCLC, we performed a genome-scale CRISPR–Cas9 sgRNA screen. We identified the deubiquitinase USP7 as a MYCN-associated synthetic vulnerability. Pharmacological inhibition of USP7 resensitized chemoresistant MYCN-overexpressing PDX models to chemotherapy in vivo. Our findings show that MYCN overexpression drives SCLC chemoresistance and provide a therapeutic strategy to restore chemosensitivity.

Epithelial ovarian cancer (EOC) is a heterogeneous group of diseases with distinct biological and clinical behaviour. Despite the differences between them, the capability of tumour cells to continuously proliferate and avoid death is maintained among histotypes. This ability is the result of alterations at different levels, causing the deregulation of cell cycle and proliferative-related pathways. Even if the leading role is played by RB and TP53, changes in other molecular pathways are involved in the development of EOC. This ability can be exploited to generate in vitro and in vivo models resembling the conditions of tumour development in a patient. In vivo models, such as patient-derived xenografts (PDX) or genetically engineered mouse models (GEMM), represent a fundamental tool in the study of the molecular mechanisms implicated in each EOC biotype for testing new therapeutic approaches. Herein we describe the major proliferation-related pathways and its disruption found in EOC and how these features can be used to establish in vivo models for translational research., Highlights • Epithelial ovarian cancer (EOC) molecular biotypes are defined by distinct biology and clinical behaviour. • Sustained proliferation and resistance to cell death mechanisms characterised tumour cells. • RB and TP53 tumour-suppressor genes are highly implicated in EOC pathogenesis. • In vitro and in vivo models have a key role in the study of molecular mechanisms involved in EOC pathogenesis. • Development of animal models that mimic disease features constitute essential tools for new drugs testing.

Peripheral neurotoxicity is a debilitating toxicity that afflicts up to 90% of patients with colorectal cancer receiving oxaliplatin-containing therapy. Although emerging evidence has highlighted the importance of various solute carriers to the toxicity of anticancer drugs, the contribution of these proteins to oxaliplatin-induced peripheral neurotoxicity remains controversial. Among candidate transporters investigated in genetically-engineered mouse models, we provide evidence for a critical role of the organic cation transporter 2 (OCT2) in satellite glial cells to oxaliplatin-induced neurotoxicity, and demonstrate that targeting OCT2 using genetic and pharmacological approaches ameliorates acute and chronic forms of neurotoxicity. The relevance of this transport system was verified in transporter-deficient rats as a secondary model organism, and translational significance of preventative strategies was demonstrated in preclinical models of colorectal cancer. These studies suggest that pharmacological targeting of OCT2 could be exploited to afford neuroprotection in cancer patients requiring treatment with oxaliplatin.

Identifying the factors stimulating prostate cancer cells migration and invasion has the potential to bring new therapeutic targets to the clinic. Cysteine-rich secretory protein 3 (CRISP3) is one of the most highly upregulated proteins during the transition of a healthy human prostatic epithelium to prostate cancer. Here we show using a genetically engineered mouse model of prostate cancer that CRISP3 production greatly facilitates disease progression from carcinoma in situ to invasive prostate cancer in vivo. This interpretation was confirmed using both human and mouse prostate cancer cell lines, which showed that exposure to CRISP3 enhanced cell motility and invasion. Further, using mass spectrometry, we show that CRISP3 induces changes in abundance of a subset of cell-cell adhesion proteins, including LASP1 and TJP1 both in vivo and in vitro. Collectively, these data identify CRISP3 as being pro-tumorigenic in the prostate and validate it as a potential target for therapeutic intervention.

Purpose of review The aim of this review is to summarize recent advances on development of in vivo preclinical models of adrenocortical carcinoma (ACC). Recent findings Significant progress has been achieved in the underlying molecular mechanisms of adrenocortical tumorigenesis over the last decade, and recent comprehensive profiling analysis of ACC tumors identified several genetic and molecular drivers of this disease. Therapeutic breakthroughs, however, have been limited because of the lack of preclinical models recapitulating the molecular features and heterogeneity of the tumors. Recent publications on genetically engineered mouse models and development of patient-derived ACC xenografts in both nude mice and humanized mice now provide researchers with novel tools to explore therapeutic targets in the context of heterogeneity and tumor microenvironment in human ACC. Summary We review current in-vivo models of ACC and discuss potential therapeutic opportunities that have emerged from these studies.

The pro-inflammatory cytokine interleukin-1 (IL-1) plays a key role in many physiological processes and during the inflammatory and immune response to most common diseases. IL-1 exists as two agonists, IL-1α and IL-1β that bind to the only signaling IL-1 type 1 receptor (IL-1R1), while a second decoy IL-1 type 2 receptor (IL-1R2) binds both forms of IL-1 without inducing cell signaling. The field of immunology and inflammation research has, over the past 35 years, unraveled many mechanisms of IL-1 actions, through in vitro manipulation of the IL-1 system or by using genetically engineered mouse models that lack either member of the IL-1 family in ubiquitous constitutive manner. However, the limitation of global mouse knockout technology has significantly hampered our understanding of the precise mechanisms of IL-1 actions in animal models of disease. Here we report and review the recent generation of new conditional mouse mutants in which exons of Il1a, Il1b, Il1r1, and Il1r2 genes flanked by loxP sites (fl/fl) can be deleted in cell-/tissue-specific constitutive or inducible manner by Cre recombinase expression. Hence, IL-1αfl/fl, IL-1βfl/fl, IL-1R1fl/fl, and IL-1R2fl/fl mice constitute a new toolbox that will provide a step change in our understanding of the cell-specific role of IL-1 and its receptor in health and disease and the potential development of targeted IL-1 therapies.

The poor prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) impels an improved understanding of disease biology to facilitate the development of better therapies. PDAC typically features a remarkably dense stromal reaction, featuring and established by a prominent population of cancer-associated fibroblasts (CAF). Genetically engineered mouse models and increasingly sophisticated cell culture techniques have demonstrated important roles for fibroblasts in PDAC progression and therapy response, but these roles are complex, with strong evidence for both tumor-supportive and tumor-suppressive or homeostatic functions. Here, we review the recent literature that has improved our understanding of heterogeneity in fibroblast fate and function in this disease including the existence of distinct fibroblast populations, and highlight important avenues for future study. Significance: Although the abundant stromal reaction associated with pancreatic cancer has long been appreciated, the functions of the CAF cells that establish this stromal reaction remain unclear. An improved understanding of the transcriptional and functional heterogeneity of pancreatic CAFs, as well as their tumor-supportive versus tumor-suppressive capacity, may facilitate the development of effective therapies for this disease.

Colorectal cancer (CRC) is the third most common diagnosed malignancy among both sexes in the United States as well as in the European Union. While the incidence and mortality rates in western, high developed countries are declining, reflecting the success of screening programs and improved treatment regimen, a rise of the overall global CRC burden can be observed due to lifestyle changes paralleling an increasing human development index. Despite a growing insight into the biology of CRC and many therapeutic improvements in the recent decades, preclinical in vivo models are still indispensable for the development of new treatment approaches. Since the development of carcinogen-induced rodent models for CRC more than 80 years ago, a plethora of animal models has been established to study colon cancer biology. Despite tenuous invasiveness and metastatic behavior, these models are useful for chemoprevention studies and to evaluate colitis-related carcinogenesis. Genetically engineered mouse models (GEMM) mirror the pathogenesis of sporadic as well as inherited CRC depending on the specific molecular pathways activated or inhibited. Although the vast majority of CRC GEMM lack invasiveness, metastasis and tumor heterogeneity, they still have proven useful for examination of the tumor microenvironment as well as systemic immune responses; thus, supporting development of new therapeutic avenues. Induction of metastatic disease by orthotopic injection of CRC cell lines is possible, but the so generated models lack genetic diversity and the number of suited cell lines is very limited. Patient-derived xenografts, in contrast, maintain the pathological and molecular characteristics of the individual patient's CRC after subcutaneous implantation into immunodeficient mice and are therefore most reliable for preclinical drug development - even in comparison to GEMM or cell line-based analyses. However, subcutaneous patient-derived xenograft models are less suitable for studying most aspects of the tumor microenvironment and anti-tumoral immune responses. The authors review the distinct mouse models of CRC with an emphasis on their clinical relevance and shed light on the latest developments in the field of preclinical CRC models.

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.

The cumbersome and time-consuming process of generating new mouse strains and multiallelic experimental animals often hinders the use of genetically engineered mouse models (GEMM) in cancer research. Here, we describe the development and validation of an embryonic stem cell (ESC)-GEMM platform for rapid modeling of melanoma in mice. The platform incorporates 12 clinically relevant genotypes composed of combinations of four driver alleles (LSL-BrafV600E, LSL-NrasQ61R, PtenFlox, and Cdkn2aFlox) and regulatory alleles to spatiotemporally control the perturbation of genes of interest. The ESCs produce high-contribution chimeras, which recapitulate the melanoma phenotypes of conventionally bred mice. Using the ESC-GEMM platform to modulate Pten expression in melanocytes in vivo, we highlighted the utility and advantages of gene depletion by CRISPR-Cas9, RNAi, or conditional knockout for melanoma modeling. Moreover, complementary genetic methods demonstrated the impact of Pten restoration on the prevention and maintenance of Pten-deficient melanomas. Finally, we showed that chimera-derived melanoma cell lines retain regulatory allele competency and are a powerful resource to complement ESC-GEMM chimera experiments in vitro and in syngeneic grafts in vivo. Thus, when combined with sophisticated genetic tools, the ESC-GEMM platform enables rapid, high-throughput, and versatile studies aimed at addressing outstanding questions in melanoma biology. Significance: This study presents a high-throughput and versatile ES cell-based mouse modeling platform that can be combined with state-of-the-art genetic tools to address unanswered questions in melanoma in vivo. See related commentary by Thorkelsson et al., p. 655

Robust preclinical models of ovarian high-grade serous carcinoma (HGSC) are needed to advance our understanding of HGSC pathogenesis and to test novel strategies aimed at improving clinical outcomes for women with the disease. Genetically engineered mouse models of HGSC recapitulating the likely cell of origin (fallopian tube), underlying genetic defects, histology, and biologic behavior of human HGSCs have been developed. However, the degree to which the mouse tumors acquire the somatic genomic changes, gene expression profiles, and immune microenvironment that characterize human HGSCs remains unclear. We used integrated molecular characterization of oviductal HGSCs arising in the context of Brca1, Trp53, Rb1, and Nf1 (BPRN) inactivation to determine whether the mouse tumors recapitulate human HGSCs across multiple domains of molecular features. Targeted DNA sequencing showed the mouse BPRN tumors, but not endometrioid carcinoma-like tumors based on different genetic defects (e.g., Apc and Pten), acquire somatic mutations and widespread copy number alterations similar to those observed in human HGSCs. RNA sequencing showed the mouse HGSCs most closely resemble the so-called immunoreactive and mesenchymal subsets of human HGSCs. A combined immuno-genomic analysis demonstrated the immune microenvironment of BPRN tumors models key aspects of tumor-immune dynamics in the immunoreactive and mesenchymal subtypes of human HGSC, with enrichment of immunosuppressive cell subsets such as myeloid-derived suppressor cells and regulatory T cells. The findings further validate the BPRN model as a robust preclinical experimental platform to address current barriers to improved prevention, diagnosis, and treatment of this often lethal cancer. Significance: The acquired gene mutations, broad genomic alterations, and gene expression and immune cell–tumor axis changes in a mouse model of oviductal serous carcinoma closely mirror those of human tubo-ovarian high-grade serous carcinoma.

The bioartificial liver (BAL) device is an extracorporeal liver support system incorporating living hepatocytes. A major problem in BAL device development is to obtain a high number of functional cells. In this study, we focused on a genetically engineered mouse hepatoma cell line, Hepa/8F5, in which elevated liver functions are induced via overexpression of liver-enriched transcription factors activated by doxycycline (Dox) addition. We applied a three-dimensional culture technique using hollow fibers (HFs) to Hepa/8F5 cells. Hepa/8F5 cells responded to Dox addition by reducing their proliferative activity and performing liver-specific functions of ammonia removal and albumin secretion. The functional activities of cells depended on the timing of Dox addition. We also found that Hepa/8F5 cells in the HF culture were highly functional in a low rather than high cell density environment. We further fabricated an HF-type bioreactor with immobilized Hepa/8F5 cells as a BAL device. Although ammonia removal activity of this BAL device was lower than that of the small-scale HF bundle, albumin secretion activity was slightly higher. These results indicated that the BAL device with immobilized Hepa/8F5 cells was highly functional with potential to show curative effects in liver failure treatment.

Informative and realistic mouse models of high-risk neuroblastoma are central to understanding mechanisms of tumour initiation, progression, and metastasis. They also play vital roles in validating tumour drivers and drug targets, as platforms for assessment of new therapies and in the generation of drug sensitivity data that can inform treatment decisions for individual patients. This review will describe genetically engineered mouse models of specific subsets of high-risk neuroblastoma, the development of patient-derived xenograft models that more broadly represent the diversity and heterogeneity of the disease, and models of primary and metastatic disease. We discuss the research applications, advantages, and limitations of each model type, the importance of model repositories and data standards for supporting reproducible, high-quality research, and potential future directions for neuroblastoma mouse models.

Mathematical models of tumor growth in response to chemotherapy are crucial for therapy optimization and outcome. We create a relatively simple tumor growth model describing the antitumor effect of pegylated liposomal doxorubicin (PLD) validated with real experimental data obtained in a genetically engineered mouse model of breast cancer. We use formal reaction kinetics to describe the pathophysiological phenomena using differential equations, and carry out parametric identification based on experiments using a mixed-effect model with stochastic approximation expectation maximization. The model gives a sufficient fit to describe tumor growth and pharmacokinetic data, and a satisfactory fit for the complex case, i.e., tumor response to chemotherapy. The results showed that identification of certain subsystems is easy using experimental data even if it is not specifically designed for identification. However, the identification of the complex pathophysiological phenomena may require experiments specially designed for identification purposes.

Gastric cancer is the third leading cause of cancer‐related mortality worldwide. This is in part due to the asymptomatic nature of the disease, which often results in late‐stage diagnosis, at which point there are limited treatment options. Even when treated successfully, gastric cancer patients have a high risk of tumor recurrence and acquired drug resistance. It is vital to gain a better understanding of the molecular mechanisms underlying gastric cancer pathogenesis to facilitate the design of new‐targeted therapies that may improve patient survival. A number of chemically and genetically engineered mouse models of gastric cancer have provided significant insight into the contribution of genetic and environmental factors to disease onset and progression. This review outlines the strengths and limitations of current mouse models of gastric cancer and their relevance to the pre‐clinical development of new therapeutics.

Cystic fibrosis mouse models are tools that enable us to study and understand the mechanisms and complexities of cystic fibrosis. Mouse models are better, and usually preferred when compared to other animal models, because of their high reproduction rate, low cost, and easy maintenance. A Mouse can be easily genetically manipulated by making it a transgenic or knockout mouse. However, as there are anatomic and immunological differences between mice and humans, these models have inherent limitations which need to be accounted for when analyzing the results obtained from experiments. This review focuses on the different CF mouse models which represent diverse phenotypes observed in humans with cystic fibrosis. This can potentially help researchers interpret the diverse functions of the CFTR protein.

Targeting the DNA damage response represents a promising approach to improve the efficacy of radiation therapy. One appealing target for this approach is the serine/threonine kinase ataxia telangiectasia mutated (ATM), which is activated by DNA double strand breaks to orchestrate the cellular response to ionizing radiation. Small-molecule inhibitors targeting ATM have entered clinical trials testing their safety in combination with radiation therapy or in combination with other DNA damaging agents. Here, we review biochemical, genetic, and cellular functional studies of ATM, phenotypes associated with germline and somatic cancer mutations in ATM in humans, and experiments in genetically engineered mouse models that support a rationale for investigating ATM inhibitors as radiosensitizers for cancer therapy. These data identify important synthetic lethal relationships, which suggest that ATM inhibitors may be particularly effective in tumors with defects in other nodes of the DNA damage response. The potential for ATM inhibition to improve immunotherapy responses in preclinical models represents another emerging area of research. We summarize ongoing clinical trials of ATM inhibitors with radiotherapy. We also discuss critical ongoing areas of investigation that include discovery of biomarkers that predict for radiosensitization by ATM inhibitors and identification of effective combinations of ATM inhibitors, radiation therapy, other DNA damage response-directed therapies, and/or immunotherapies.

Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological interventions, we describe an unexpected dual role of the tumor necrosis factor receptor–associated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.

Neuroblastoma, the most common extracranial solid tumor in young children, arises from the sympathetic nervous system. Our understanding of neuroblastoma has been improved by the development of both genetically engineered and xenograft mouse models of the disease. Anatomical pathology is an essential component of the phenotyping of mouse models of cancer, characterizing the morphologic effects of genetic manipulation and drug treatment. The Th-MYCN model, the most widely used of several genetically engineered mouse models of neuroblastoma, was established by targeted expression of the human MYCN gene to murine neural crest cells under the control of the rat tyrosine hydroxylase promoter. Neuroblastoma development in Th-MYCN mice is preceded by neuroblast hyperplasia-the persistence and proliferation of neural crest-derived neuroblasts within the sympathetic autonomic ganglia. The neuroblastomas that subsequently develop morphologically resemble human neuroblastoma and carry chromosomal gains and losses in regions syntenic with those observed in human tumors. In this overview, we describe the essential pathologic features for investigators when assessing mouse models of neuroblastoma. We outline human neuroblastoma as the foundation for understanding the murine disease, followed by details of the murine sympathetic ganglia from which neuroblastoma arises. Sympathetic ganglia, both with and without neuroblast hyperplasia, are described. The macroscopic and microscopic features of murine neuroblastoma are explained, including assessment of xenografts and tumors following drug treatment. An approach to experimental design is also detailed. Increased understanding of the pathology of murine neuroblastoma should improve reproducibility and comparability of research findings and assist investigators working with mouse models of neuroblastoma. © 2021 Wiley Periodicals LLC.

The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. Large-scale comparison of fertilization rates from independent lines of genetically engineered C57BL/6 mice upon cryopreservation and in vitro fertilization with our approach demonstrated equal or significantly superior fertilization rates to current protocols. Our novel SEcuRe (Simple Economical set-up for Rederivation) method provides an affordable, easily adaptable and harmonized protocol for highly efficient rederivation using cryopreserved C57BL/6 sperm for a broad application of colony management in the sense of the 3Rs.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide, and survival rates have barely improved in decades. In the era of precision medicine, treatment strategies tailored to disease mutations have revolutionized cancer therapy. Next generation sequencing has found that up to a third of all PDAC tumors contain deleterious mutations in DNA damage repair (DDR) genes, highlighting the importance of these genes in PDAC. The mechanisms by which DDR gene mutations promote tumorigenesis, therapeutic response, and subsequent resistance are still not fully understood. Therefore, an opportunity exists to elucidate these processes and to uncover relevant therapeutic drug combinations and strategies to target DDR deficiency in PDAC. However, a constraint to preclinical research is due to limitations in appropriate laboratory experimental models. Models that effectively recapitulate their original cancer tend to provide high levels of predictivity and effective translation of preclinical findings to the clinic. In this review, we outline the occurrence and role of DDR deficiency in PDAC and provide an overview of clinical trials that target these pathways and the preclinical models such as 2D cell lines, 3D organoids and mouse models [genetically engineered mouse model (GEMM), and patient-derived xenograft (PDX)] used in PDAC DDR deficiency research.

Although many cancer prognoses have improved in the past 50 years due to advancements in treatments, there has been little improvement in therapies for small-cell lung cancer (SCLC). One promising avenue to improve treatment for SCLC is to understand its underlying genetic alterations that drive its formation, growth, and cellular heterogeneity. RB1 loss is one key driver of SCLC, and RB1 loss has been associated with an increase in pluripotency factors such as SOX2. SOX2 is highly expressed and amplified in SCLC and has been associated with SCLC growth. Using a genetically engineered mouse model, we have shown that Sox2 is required for efficient SCLC formation. Furthermore, genome-scale binding assays have indicated that SOX2 can regulate key SCLC pathways such as NEUROD1 and MYC. These data suggest that SOX2 can be associated with the switch of SCLC from an ASCL1 subtype to a NEUROD1 subtype. Understanding this genetic switch is key to understanding such processes as SCLC progression, cellular heterogeneity, and treatment resistance. Implications: Understanding the molecular mechanisms of SCLC initiation and development are key to opening new potential therapeutic options for this devastating disease.

Tyr::CreER+,BrafCA/+,Ptenlox/lox genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume. The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 µm). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 µm step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement. This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Glioblastoma (GBM) is an incurable brain tumor with a median survival of approximately 15 months despite an aggressive standard of care that includes surgery, chemotherapy, and ionizing radiation. Mouse models have advanced our understanding of GBM biology and the development of novel therapeutic strategies for GBM patients. However, model selection is crucial when testing developmental therapeutics, and each mouse model of GBM has unique advantages and disadvantages that can influence the validity and translatability of experimental results. To shed light on this process, we discuss the strengths and limitations of 3 types of mouse GBM models in this review: syngeneic models, genetically engineered mouse models, and xenograft models, including traditional xenograft cell lines and patient-derived xenograft models.