Genetically engineered mouse model of HPV16 E6-E7 with vaginal-cervical intraepithelial neoplasia and decreased immunity.

Objective: To construct models of high-risk human papillomavirus (HPV) infection with precancerous lesions or cervical cancer and explore the immune function. Methods: Using CRISPR/Cas9, the expression vector HPV16-E6-E7-Rosa26 was microinjected into fertilized eggs of C57BL/6 N mice using homologous recombination, and the F0 generation was obtained for reproduction. Then, the formation of precancerous lesions was promoted via intramuscular injection of estradiol. Presence of precancerous cervical-vaginal intraepithelial lesions, Ki67 and p16 expression levels, and CD8+ T cell proportions in the spleen were evaluated. Results: Two F0 generation mice exhibited correct the homologous recombination. Seven positive mice were identified in the F1 generation. After breeding and mating, 25 homozygous and 11 heterozygous HPV16-E6-E7-engineered mice were obtained from the F2 generation. After estradiol benzoate treatment, the cervical-vaginal epithelium appeared as precancerous lesions with positive Ki67 and p16 expression. The percentage of CD8+ T cells decreased. Conclusion: HPV16-E6-E7-Rosa26 induced low immune function in mice, and provides a good model for the basic research of the mechanisms of action of HPV infection-associated precancerous lesions or cervical cancer.

Optimizing preclinical pediatric low-grade glioma models for meaningful clinical translation.

Pediatric low-grade gliomas (pLGGs) are the most common brain tumor in young children. While they are typically associated with good overall survival, children with these central nervous system tumors often experience chronic tumor- and therapy-related morbidities. Moreover, individuals with unresectable tumors frequently have multiple recurrences and persistent neurological symptoms. Deep molecular analyses of pLGGs reveal that they are caused by genetic alterations that converge on a single mitogenic pathway (MEK/ERK), but their growth is heavily influenced by nonneoplastic cells (neurons, T cells, microglia) in their local microenvironment. The interplay between neoplastic cell MEK/ERK pathway activation and stromal cell support necessitates the use of predictive preclinical models to identify the most promising drug candidates for clinical evaluation. As part of a series of white papers focused on pLGGs, we discuss the current status of preclinical pLGG modeling, with the goal of improving clinical translation for children with these common brain tumors.

Mouse models for immuno-oncology.

Realizing the clinical promise of cancer immunotherapy is hindered by gaps in our knowledge of in vivo mechanisms underlying treatment response as well as treatment limiting toxicity. Preclinical in vivo model systems and technologies are required to address these knowledge gaps and to surmount the challenges faced in the clinical application of immunotherapy. Mice are commonly used for basic and translational research to support development and testing of immune interventions, including for cancer. Here, we discuss the advantages and the limitations of current models as well as future developments.

Hypoxia-inducible factor 1A inhibition overcomes castration resistance of prostate tumors.

Androgen deprivation therapy (ADT) is a cornerstone of prostate cancer (PCa) management. Although tumors initially regress, many progress to a hormone-independent state termed castration-resistant PCa (CRPC), for which treatment options are limited. We here report that the major luminal cell population in tumors of Pten(i)pe-/- mice, generated by luminal epithelial cell-specific deletion of the tumor suppressor PTEN after puberty, is castration-resistant and that the expression of inflammation and stemness markers is enhanced in persistent luminal cells. In addition, hypoxia-inducible factor 1 (HIF1) signaling, which we have previously demonstrated to be induced in luminal cells of Pten(i)pe-/- mice and to promote malignant progression, is further activated. Importantly, we show that genetic and pharmacological inhibition of HIF1A sensitizes Pten-deficient prostatic tumors to castration and provides durable therapeutic responses. Furthermore, HIF1A inhibition induces apoptotic signaling in human CRPC cell lines. Therefore, our data demonstrate that HIF1A in prostatic tumor cells is a critical factor that enables their survival after ADT, and identify it as a target for CRPC management.

Neurofibromatosis type 1-dependent alterations in mouse microglia function are not cell-intrinsic.

We previously discovered a sex-by-genotype defect in microglia function using a heterozygous germline knockout mouse model of Neurofibromatosis type 1 (Nf1 ± mice), in which only microglia from male Nf1 ± mice exhibited defects in purinergic signaling. Herein, we leveraged an unbiased proteomic approach to demonstrate that male, but not female, heterozygous Nf1 ± microglia exhibit differences in protein expression, which largely reflect pathways involved in cytoskeletal organization. In keeping with these predicted defects in cytoskeletal function, only male Nf1 ± microglia had reduced process arborization and surveillance capacity. To determine whether these microglial defects were cell autonomous or reflected adaptive responses to Nf1 heterozygosity in other cells in the brain, we generated conditional microglia Nf1-mutant knockout mice by intercrossing Nf1flox/flox with Cx3cr1-CreER mice (Nf1flox/wt; Cx3cr1-CreER mice, Nf1MG ± mice). Surprisingly, neither male nor female Nf1MG ± mouse microglia had impaired process arborization or surveillance capacity. In contrast, when Nf1 heterozygosity was generated in neurons, astrocytes and oligodendrocytes by intercrossing Nf1flox/flox with hGFAP-Cre mice (Nf1flox/wt; hGFAP-Cre mice, Nf1GFAP ± mice), the microglia defects found in Nf1 ± mice were recapitulated. Collectively, these data reveal that Nf1 ± sexually dimorphic microglia abnormalities are likely not cell-intrinsic properties, but rather reflect a response to Nf1 heterozygosity in other brain cells.

Volumetric imaging of optically cleared and fluorescently labeled animal tissue (VIOLA) for quantifying the 3D biodistribution of nanoparticles at cellular resolution in tumor tissue.

Nanoparticle (NP) technology holds significant promise to mediate targeted drug delivery to specific organs in the body. Understanding the 3D biodistribution of NPs in heterogeneous environments such as the tumor tissue can provide crucial information on efficacy, safety and potential clinical outcomes. Here we present a novel end-to-end workflow, VIOLA, which makes use of tissue clearing methodology in conjunction with high resolution imaging and advanced 3D image processing to quantify the spatiotemporal 3D biodistribution of fluorescently labeled ACCURIN® NPs. Specifically, we investigate the spatiotemporal biodistribution of NPs in three different murine tumor models (CT26, EMT6, and KPC-GEM) of increasing complexity and translational relevance. We have developed new endpoints to characterize NP biodistribution at multiple length scales. Our observations reveal that the macroscale NP biodistribution is spatially heterogeneous and exhibits a gradient with relatively high accumulation at the tumor periphery that progressively decreases towards the tumor core in all the tumor models. Microscale analysis revealed that NP extravasation from blood vessels increases in a time dependent manner and plateaus at 72 h post injection. Volumetric analysis and pharmacokinetic modeling of NP biodistribution in the vicinity of the blood vessels revealed that the local NP density exhibits a distance dependent spatiotemporal biodistribution which provide insights into the dynamics of NP extravasation in the tumor tissue. Our data represents a comprehensive analysis of NP biodistribution at multiple length scales in different tumor models providing unique insights into their spatiotemporal dynamics. Specifically, our results show that NPs exhibit a dynamic equilibrium with macroscale heterogeneity combined with microscale homogeneity.

Genetically engineered mouse models for hereditary cancer syndromes.

Advances in molecular diagnostics have led to improved diagnosis and molecular understanding of hereditary cancers in the clinic. Improving the management, treatment, and potential prevention of cancers in carriers of predisposing mutations requires preclinical experimental models that reflect the key pathogenic features of the specific syndrome associated with the mutations. Numerous genetically engineered mouse (GEM) models of hereditary cancer have been developed. In this review, we describe the models of Lynch syndrome and hereditary breast and ovarian cancer syndrome, the two most common hereditary cancer predisposition syndromes. We focus on Lynch syndrome models as illustrative of the potential for using mouse models to devise improved approaches to prevention of cancer in a high-risk population. GEM models are an invaluable tool for hereditary cancer models. Here, we review GEM models for some hereditary cancers and their potential use in cancer prevention studies.

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System.

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

Generation of genetically engineered mice for lung cancer with mutant EGFR.

Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have shown dramatic response and improvement in treating lung cancer with mutant EGFR, the emergence of drug resistance remains a major problem. In particular, some mutations including T790 M and C797S have been recognized as mechanisms of acquired resistance because they weaken binding affinity to drugs. To date, many attempts have been made to develop a new drug for overcoming acquired resistance to EGFR-TKIs, including secondary mutations. However, an appropriate animal model to evaluate in vivo efficacy during novel drug development remains lacking. In this study, we generated a novel transgenic mouse model that conditionally expresses human EGFRL858R/T790M/C797S and firefly luciferase using Cas9-mediated homology-independent targeted integration. Using a lung-specific Sftpc-CreERT2 mouse line, we induced expression of both the human EGFRL858R/T790M/C797S transgene and firefly luciferase in the lungs of adult mice. The expression of these genes and lung cancer occurrence was monitored using an in vivo imaging system and magnetic resonance imaging, respectively. Overall, our mouse model can be utilized to develop new drugs for overcoming C797S-mediated resistance to osimertinib; further, such knock-in systems for expressing oncogenes may be applied to study tumorigenesis and the development of other targeted agents.

Genetically manipulating endogenous Kras levels and oncogenic mutations in vivo influences tissue patterning of murine tumorigenesis.

Despite multiple possible oncogenic mutations in the proto-oncogene KRAS, unique subsets of these mutations are detected in different cancer types. As KRAS mutations occur early, if not being the initiating event, these mutational biases are ostensibly a product of how normal cells respond to the encoded oncoprotein. Oncogenic mutations can impact not only the level of active oncoprotein, but also engagement with proteins. To attempt to separate these two effects, we generated four novel Cre-inducible (LSL) Kras alleles in mice with the biochemically distinct G12D or Q61R mutations and encoded by native (nat) rare or common (com) codons to produce low or high protein levels. While there were similarities, each allele also induced a distinct transcriptional response shortly after activation in vivo. At one end of the spectrum, activating the KrasLSL-natG12D allele induced transcriptional hallmarks suggestive of an expansion of multipotent cells, while at the other end, activating the KrasLSL-comQ61R allele led to hallmarks of hyperproliferation and oncogenic stress. Evidence suggests that these changes may be a product of signaling differences due to increased protein expression as well as the specific mutation. To determine the impact of these distinct responses on RAS mutational patterning in vivo, all four alleles were globally activated, revealing that hematolymphopoietic lesions were permissive to the level of active oncoprotein, squamous tumors were permissive to the G12D mutant, while carcinomas were permissive to both these features. We suggest that different KRAS mutations impart unique signaling properties that are preferentially capable of inducing tumor initiation in a distinct cell-specific manner.

Making a mouse out of a molehill: how precision modeling repurposes drugs for congenital giant nevi.

Patients with congenital giant nevi (CGN), which can compromise quality of life and progress to melanoma, have limited treatment options. Choi et al. have demonstrated that topical application of a proinflammatory hapten for alopecia treatment [squaric acid dibutylester (SADBE)] caused nevus regression and prevented melanoma in an Nras mouse CGN model. Their results demonstrate the promise of repurposing drugs through precision modeling.

Murine Models of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a phenotypically and genetically heterogeneous hematologic malignancy. Extensive sequencing efforts have mapped the genomic landscape of adult and pediatric AML revealing a number of biologically and prognostically relevant driver lesions. Beyond identifying recurrent genetic aberrations, it is of critical importance to fully delineate the complex mechanisms by which they contribute to the initiation and evolution of disease to ultimately facilitate the development of targeted therapies. Towards these aims, murine models of AML are indispensable research tools. The rapid evolution of genetic engineering techniques over the past 20 years has greatly advanced the use of murine models to mirror specific genetic subtypes of human AML, define cell-intrinsic and extrinsic disease mechanisms, study the interaction between co-occurring genetic lesions, and test novel therapeutic approaches. This review summarizes the mouse model systems that have been developed to recapitulate the most common genomic subtypes of AML. We will discuss the strengths and weaknesses of varying modeling strategies, highlight major discoveries emanating from these model systems, and outline future opportunities to leverage emerging technologies for mechanistic and preclinical investigations.

Receptor Activity Modifying Protein RAMP Sub-Isoforms and Their Functional Differentiation, Which Regulates Functional Diversity of Adrenomedullin.

AM knockout (AM-/-) and RAMP2 knockout (RAMP2-/-) proved lethal for mice due to impaired embryonic vascular development. Although most vascular endothelial cell-specific RAMP2 knockout (E-RAMP2-/-) mice also died during the perinatal period, a few E-RAMP2-/- mice reached adulthood. Adult E-RAMP2-/- mice developed spontaneous organ damage associated with vascular injury. In contrast, adult RAMP3 knockout (RAMP3-/-) mice showed exacerbated postoperative lymphedema with abnormal lymphatic drainage. Thus, RAMP2 is essential for vascular development and homeostasis and RAMP3 is essential for lymphatic vessel function. Cardiac myocyte-specific RAMP2 knockout mice showed early onset of heart failure as well as abnormal mitochondrial morphology and function, whereas RAMP3-/- mice exhibited abnormal cardiac lymphatics and a delayed onset of heart failure. Thus, RAMP2 is essential for maintaining cardiac mitochondrial function, while RAMP3 is essential for cardiac lymphangiogenesis. Transplantation of cancer cells into drug-inducible vascular endothelial cell-specific RAMP2 knockout mice resulted in enhanced metastasis to distant organs, whereas metastasis was suppressed in RAMP3-/- mice. RAMP2 suppresses cancer metastasis by maintaining vascular homeostasis and inhibiting vascular inflammation and pre-metastatic niche formation, while RAMP3 promotes cancer metastasis via malignant transformation of cancer-associated fibroblasts. Focusing on the diverse physiological functions of AM and the functional differentiation of RAMP2 and RAMP3 may lead to the development of novel therapeutic strategies.

Cell Type-Specific Genetic Manipulation and Impaired Circadian Rhythms in Vip tTA Knock-In Mice.

The suprachiasmatic nucleus (SCN), the central circadian clock in mammals, is a neural network consisting of various types of GABAergic neurons, which can be differentiated by the co-expression of specific peptides such as vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP). VIP has been considered as a critical factor for the circadian rhythmicity and synchronization of individual SCN neurons. However, the precise mechanisms of how VIP neurons regulate SCN circuits remain incompletely understood. Here, we generated Vip tTA knock-in mice that express tetracycline transactivator (tTA) specifically in VIP neurons by inserting tTA sequence at the start codon of Vip gene. The specific and efficient expression of tTA in VIP neurons was verified using EGFP reporter mice. In addition, combined with Avp-Cre mice, Vip tTA mice enabled us to simultaneously apply different genetic manipulations to VIP and AVP neurons in the SCN. Immunostaining showed that VIP is expressed at a slightly reduced level in heterozygous Vip tTA mice but is completely absent in homozygous mice. Consistently, homozygous Vip tTA mice showed impaired circadian behavioral rhythms similar to those of Vip knockout mice, such as attenuated rhythmicity and shortened circadian period. In contrast, heterozygous mice demonstrated normal circadian behavioral rhythms comparable to wild-type mice. These data suggest that Vip tTA mice are a valuable genetic tool to express exogenous genes specifically in VIP neurons in both normal and VIP-deficient mice, facilitating the study of VIP neuronal roles in the SCN neural network.

Effects of hyperinsulinemia on pancreatic cancer development and the immune microenvironment revealed through single-cell transcriptomics.

BACKGROUND: Hyperinsulinemia is independently associated with increased risk and mortality of pancreatic cancer. We recently reported that genetically reduced insulin production resulted in ~ 50% suppression of pancreatic intraepithelial neoplasia (PanIN) precancerous lesions in mice. However, only female mice remained normoglycemic, and only the gene dosage of the rodent-specific Ins1 alleles was tested in our previous model. Moreover, we did not delve into the molecular and cellular mechanisms associated with modulating hyperinsulinemia. METHODS: We studied how reduced Ins2 gene dosage affects PanIN lesion development in both male and female Ptf1aCreER;KrasLSL-G12D mice lacking the rodent-specific Ins1 gene (Ins1-/-). We generated control mice having two alleles of the wild-type Ins2 gene (Ptf1aCreER;KrasLSL-G12D;Ins1-/-;Ins2+/+) and experimental mice having one allele of Ins2 gene (Ptf1aCreER;KrasLSL-G12D;Ins1-/-;Ins2+/-). We then performed thorough histopathological analyses and single-cell transcriptomics for both genotypes and sexes. RESULTS: High-fat diet-induced hyperinsulinemia was transiently or modestly reduced in female and male mice, respectively, with only one allele of Ins2. This occurred without dramatically affecting glucose tolerance. Genetic reduction of insulin production resulted in mice with a tendency for less PanIN and acinar-to-ductal metaplasia (ADM) lesions. Using single-cell transcriptomics, we found hyperinsulinemia affected multiple cell types in the pancreas, with the most statistically significant effects on local immune cell types that were highly represented in our sampled cell population. Specifically, hyperinsulinemia modulated pathways associated with protein translation, MAPK-ERK signaling, and PI3K-AKT signaling, which were changed in epithelial cells and subsets of immune cells. CONCLUSIONS: These data suggest a potential role for the immune microenvironment in hyperinsulinemia-driven PanIN development. Together with our previous work, we propose that mild suppression of insulin levels may be useful in preventing pancreatic cancer by acting on multiple cell types.

Heat shock factor 1 inhibition sensitizes pancreatic cancer to gemcitabine via the suppression of cancer stem cell-like properties.

Pancreatic cancer is a fatal disease with poor prognosis. Gemcitabine has been regarded as the mainstay of chemotherapy for pancreatic cancer; however, it is accompanied with a high rate of chemoresistance. Cancer stem cells (CSCs) are characterized by resistance to traditional chemo- and radiotherapies. We have previously reported that heat shock factor 1 (HSF1) is involved in the invasion and metastasis of pancreatic cancer, a highly conserved transcriptional factor that mediates the canonical proteotoxic stress response. Here, we investigate whether HSF1 contributes to the chemoresistance of pancreatic cancer cells caused by gemcitabine and explore the underlying mechanisms. Genetically engineered mice (LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre mice), which spontaneously develop pancreatic cancer, were used to examine the sensitivity of pancreatic cancer to gemcitabine in vivo. We found that HSF1 was enriched in sphere-forming cancer cells. Panc-1 and MiaPaCa-2 cells treated chronically with gemcitabine displayed increased transcription and expression of CSC-associated markers. In addition, gemcitabine-surviving Panc-1 and MiaPaCa-2 cells showed an increased ability to form tumorspheres. Moreover, we observed that gemcitabine treatment increased the activity and expression of HSF1, as well as transcription of its downstream targets. Finally, HSF1 inhibition significantly suppressed the expression of CSC-associated markers, augmented the cancer-killing property of gemcitabine, and increased chemosensitivity to gemcitabine in vivo. Our study reveals a novel mechanism in which HSF1 promotes the chemoresistance of pancreatic cancer to gemcitabine by modulating CSC-like properties. Targeting HSF1 could be thus a rational strategy to improve treatment outcomes.

Corrigendum: Murine models of acute myeloid leukemia.

[This corrects the article DOI: 10.3389/fonc.2022.854973.].

Model Systems for the Study of Malignant Melanoma.

Since the first resection of melanoma by Hunter in 1787, efforts to treat patients with this deadly malignancy have been ongoing. Initial work to understand melanoma biology for therapeutics development began with the employment of isolated cancer cells grown in cell cultures. However, these models lack in vivo interactions with the tumor microenvironment. Melanoma cell line transplantation into suitable animals such as mice has been informative and useful for testing therapeutics as a preclinical model. Injection of freshly isolated patient melanomas into immunodeficient animals has shown the capacity to retain the genetic heterogeneity of the tumors, which is lost during the long-term culture of melanoma cells. Upon advancement of technology, genetically engineered animals have been generated to study the spontaneous development of melanomas in light of newly discovered genetic aberrations associated with melanoma formation. Culturing melanoma cells in a matrix generate tumor spheroids, providing an in vitro environment that promotes the heterogeneity commonplace with human melanoma and displaces the need for animal care facilities. Advanced 3D cultures have been created simulating the structure and cellularity of human skin to permit in vitro testing of therapeutics on melanomas expressing the same phenotype as demonstrated in vivo. This review will discuss these models and their relevance to the study of melanomagenesis, growth, metastasis, and therapy.

In Vitro and In Vivo Tumor Models for the Evaluation of Anticancer Nanoparticles.

Multiple studies about tumor biology have revealed the determinant role of the tumor microenvironment in cancer progression, resulting from the dynamic interactions between tumor cells and surrounding stromal cells within the extracellular matrix. This malignant microenvironment highly impacts the efficacy of anticancer nanoparticles by displaying drug resistance mechanisms, as well as intrinsic physical and biochemical barriers, which hamper their intratumoral accumulation and biological activity.Currently, two-dimensional cell cultures are used as the initial screening method in vitro for testing cytotoxic nanocarriers. However, this fails to mimic the tumor heterogeneity, as well as the three-dimensional tumor architecture and pathophysiological barriers, leading to an inaccurate pharmacological evaluation.Biomimetic 3D in vitro tumor models, on the other hand, are emerging as promising tools for more accurately assessing nanoparticle activity, owing to their ability to recapitulate certain features of the tumor microenvironment and thus provide mechanistic insights into nanocarrier intratumoral penetration and diffusion rates.Notwithstanding, in vivo validation of nanomedicines remains irreplaceable at the preclinical stage, and a vast variety of more advanced in vivo tumor models is currently available. Such complex animal models (e.g., genetically engineered mice and patient-derived xenografts) are capable of better predicting nanocarrier clinical efficiency, as they closely resemble the heterogeneity of the human tumor microenvironment.Herein, the development of physiologically more relevant in vitro and in vivo tumor models for the preclinical evaluation of anticancer nanoparticles will be discussed, as well as the current limitations and future challenges in clinical translation.

Challenges and Opportunities for the Veterinary Pathologist in Biomedical Research.

Animal models have critical roles in biomedical research in promoting understanding of human disease and facilitating development of new therapies and diagnostic techniques to improve human and animal health. In the study of myriad human conditions, each model requires in-depth characterization of its assets and limitations in order for it to be used to greatest advantage. Veterinary pathology expertise is critical in understanding the relevance and translational validity of animal models to conditions under study, assessing morbidity and mortality, and validating outcomes as relevant or not to the study interventions. Clear communication with investigators and education of research personnel on the use and interpretation of pathology endpoints in animal models are critical to the success of any research program. The veterinary pathologist is underutilized in biomedical research due to many factors including misconceptions about high fiscal costs, lack of perceived value, limited recognition of their expertise, and the generally low number of veterinary pathologists currently employed in biomedical research. As members of the multidisciplinary research team, veterinary pathologists have an important role to educate scientists, ensure accurate interpretation of pathology data, maximize rigor, and ensure reproducibility to provide the most reliable data for animal models in biomedical research.