Preclinical Mouse Model Research Articles

Allogeneic CD33-directed CAR-NKT cells for the treatment of bone marrow-resident myeloid malignancies

Chimeric antigen receptor (CAR)-engineered T cell therapy holds promise for treating myeloid malignancies, but challenges remain in bone marrow (BM) infiltration and targeting BM-resident malignant cells. Current autologous CAR-T therapies also face manufacturing and patient selection issues, underscoring the need for off-the-shelf products. In this study, we characterize primary patient samples and identify a unique therapeutic opportunity for CAR-engineered invariant natural killer T (CAR-NKT) cells. Using stem cell gene engineering and a clinically guided culture method, we generate allogeneic CD33-directed CAR-NKT cells with high yield, purity, and robustness. In preclinical mouse models, CAR-NKT cells exhibit strong BM homing and effectively target BM-resident malignant blast cells, including CD33-low/negative leukemia stem and progenitor cells. Furthermore, CAR-NKT cells synergize with hypomethylating agents, enhancing tumor-killing efficacy. These cells also show minimal off-tumor toxicity, reduced graft-versus-host disease and cytokine release syndrome risks, and resistance to allorejection, highlighting their substantial therapeutic potential for treating myeloid malignancies.

Dimethyl fumarate is repurposed to ameliorate aortic aneurysm and dissection in mice.

Aortic aneurysm and dissection pose fatal threats but no effective drug therapies are available. Previous work has been directed to reduce risk factors or target key pathological events, but none of the translational efforts succeeds. Here, we attempt to repurpose dimethyl fumarate (DMF), an FDA-approved immunomodulatory drug for multiple sclerosis, for the treatment of aortic aneurysm and dissection. In three preclinical mouse models of abdominal aortic aneurysm (porcine pancreatic elastase perfusion or CaCl2 incubation) and thoracic aortic aneurysm and dissection (β-Aminopropionitrile feeding), DMF invariably protected mice from aneurysm growth, aortic dissection, rupture and death. Histological H&E and EVG staining demonstrated aortic architecture-preserving effects of DMF. Through transcriptome profiling and the connectivity map (CMap), we showed that DMF restored SRC-FAK signaling in aortic smooth muscle cells and increased collagen I turnover in the tunica media. Our work suggests the potential of DMF being repurposed for aortic aneurysm and dissection, and highlights the importance of SRC-FAK signaling in aortic homeostasis.

Advancing Alzheimer's disease pharmacotherapy: efficacy of glucocorticoid modulation with dazucorilant (CORT113176) in preclinical mouse models.

Exposure to chronic stress and high levels of glucocorticoid hormones in adulthood has been associated with cognitive deficits and increased risk of Alzheimer's disease (AD). Dazucorilant has recently emerged as a selective glucocorticoid receptor (NR3C1) modulator, exhibiting efficacy in counteracting amyloid-β toxicity in an acute model of AD. We aim to assess the therapeutic potential of dazucorilant in reversing amyloid and tau pathologies through the inhibition of glucocorticoid receptor pathological activity, and providing additional evidence for its consideration in AD treatment. The efficacy of dazucorilant was evaluated in two transgenic mouse models of amyloid pathology. The slowly progressing J20 and the aggressively pathological 5xFAD mice. Behavioural analysis was conducted to evaluate welfare, cognitive performances and anxiety levels. The activity of the glucocorticoid receptor system, neuroinflammation, amyloid burden and tau phosphorylation were examined in hippocampi. In both AD models, chronic treatment with dazucorilant improved working and long-term spatial memories along with the inhibition of glucocorticoid receptor-dependent pathogenic processes and the normalization of plasma glucocorticoid levels. Dazucorilant treatment also resulted in a reduction in tau hyperphosphorylation and amyloid production and aggregation. Additionally, dazucorilant seemed to mediate a specific re-localization of activated glial cells onto amyloid plaques in J20 mice, suggesting a restoration of physiological neuroinflammatory processes. Dazucorilant exhibited sustained disease-modifying effects in two AD models. Given that this compound has demonstrated safety and tolerability in human subjects, our results provide pre-clinical support for conducting clinical trials to evaluate its potential in AD.

The role of cGAS-STING pathway in the development of radiation-induced lung injury

Background and purposeRadiation-induced lung injury (RILI) limits the efficacy of thoracic radiotherapy. However, the underlying mechanism of RILI remains unclear. cGAS-STING pathway is reported to be involved in the recognization of cytosolic dsDNA and various inflammatory diseases. This study aimed to investigate the role of cGAS-STING pathway in the development of RILI.Materials and methodsA pre-clinical mouse model of RILI was established by whole thorax irradiation and confirmed using H&E and Masson’s trichrome staining. STING agonist (DMXAA) and antagonist(C-176) were administrated to modulate cGAS-STING pathway in vivo. Western blot and ELISA were used to determine the expression levels of different proteins.ResultsQuantitation analysis showed dsDNA accumulation in lung tissue and western blot showed the up-regulation of cGAS and STING protein level post-irradiation, indicating pathway activation. Histological evaluation showed that C-176 administration ameliorated radiation-induced pulmonary inflammation and fibrosis, while DMXAA exhibited contrary effects. In further in vitro study, the release of dsDNA induced by radiation led to the activation of cGAS-STING pathway in RAW 264.7 cells, resulting in the polarization into M1 phenotype and pro-inflammatory production.ConclusionIn summary, our data demonstrated a link between cGAS-STING pathway and the development of RILI, indicating its potential application in clinic.

P0202 Modeling Inflammatory Bowel Disease in mice with “Humanized” immune system

Abstract Background Inflammatory bowel disease (IBD) is a chronic recurrent inflammatory disease with unknown etiology. Dextran sulfate sodium (DSS) induced colitis is a common preclinical mouse model in IBD research. DSS colitis involves activation of the submucosal immune system and can be used to study Ulcerative Colitis characteristics in different phases (acute, chronic and remission) and predict compound efficacy. However, optimizing translation to human is still needed and the use of mice with Human Immune System (HIS) provide a better model to study human immune system responses in vivo. HIS mice are created by transplanting human immune cells or their progenitor cells into highly immunodeficient recipient mouse hosts, thereby "humanizing" their immune systems. In this study we discussed colitis feature and the use of humanized mice model for testing therapeutics in preclinical studies Methods 15-weeks-old female HIS mice were treated with 2.5% dextran sulfate sodium (DSS) in drinking water (ad.lib.) for 5 days to induce acute colitis. Colonic and systemic human cytokines were assessed by MSD plex assay. Human immune cell infiltration into the colon was measured by flow cytometry. Colonic histological damage was assessed on colon Swiss roll after H&E staining Results HIS mice challenged with DSS exhibited body weight loss and increase disease activity index (loose stool or diarrhea, rectal bleeding) compared to naive HIS mice. HIS-DSS mice show a strong human pro-inflammatory response, including elevated hIL-1B, hTNF, hIL-6 in plasma and colon as well as specific human cytokines (MCP-4, IL-8, IL-26, IL-37…). Colon microscopic analysis show loss of goblet cells, loss of crypt structure and increase of inflammatory cell infiltration at the level of mucosa and submucosa Conclusion These data show a significant increase of human pro-inflammatory cytokines after DSS intake as well as histologic score characteristic of Ulcerative Colitis. They also confirm that HIS mice represent a relevant model for the preclinical assessment of therapeutics in IBD.

Evaluation of host immune responses to Mycobacteriophage Fionnbharth by route of delivery

For much of the last decade, tuberculosis (TB) was the leading cause of mortality due to an infectious pathogen (Mycobacterium tuberculosis, M.tb). Approximately 1.3 million deaths in 2023 worldwide were attributed to TB disease. Focused intervention strategies to block transmission would significantly reduce the global health burden of TB. Mycobacteriophages (phages) are a sorely underutilized biologic therapy for the pathogen M.tb, and here we aimed to address outstanding questions about their utility for clinical applications. We aimed to determine the impact of repeated mucosal or intravenous (IV) delivery of representative anti-M.tb phage FionnbharthΔ45Δ47 (Fionnbharth) in a preclinical mouse model. In addition, we specifically sought to understand which route induced anti-phage antibodies, which may reduce the long-term impact of phage therapy. C57BL/6 mice were dosed weekly for 6 weeks by either route and serum and bronchoalveolar lavage fluid (BALf) were evaluated for anti-phage humoral responses by ELISA. We found that aerosol delivery disperses phage across all lung lobes where M.tb is also found after experimental infection by the same route. Repeated aerosol delivery was well tolerated and did not induce robust neutralizing humoral immunity. In contrast, Mice receiving IV phage developed increasing magnitude and neutralizing total IgG and IgA responses over time. To determine whether pre-treatment environmental exposure to Fionnbharth-like phages could induce antibody responses that are potentially neutralizing, ~ 500 human plasma samples from normal donors were evaluated by ELISA. We observed that 5% of samples had antibodies to Fionnbharth (with end point titers > 10− 3 dilution), although none were neutralizing. Furthermore, we found that highly-purified phage preparations did not activate mouse or human derived toll like receptor (TLR) 4 or TLR9 in HEKblue reporter assays. These data together support using Fionnbharth in anti-M.tb therapy phage cocktail strategies and that aerosol delivery should be prioritized for further efficacy testing.

Mice develop obesity and lose myocardial metabolic flexibility months after exertional heat stroke

As global temperatures rise, heat-related chronic health disorders are predicted to become more prevalent. We tested whether a single exposure to acute heat illness, using a preclinical mouse model of exertional heat stroke (EHS), can induce late-emerging health disorders that progress into chronic disease. Following EHS, mice were followed for 3 months; after two weeks of recovery, half were placed on a Western diet to determine if previous EHS exposure amplifies the negative consequences of an atherogenic diet. When compared to sham exercise controls, EHS-exposed mice exhibit accelerated diet-induced obesity, develop low level cardiac hypertrophy, develop accelerated diet-induced liver steatosis, severe hypoproteinemia and a loss of metabolic flexibility in the myocardium. The latter is characterized by a shift towards predominant glucose metabolism and glycolysis. These results demonstrate that a single exposure to severe exertional heat illness can induce long-lasting and unexpected health consequences in mammals and increased vulnerability to secondary metabolic stressors.

Update on Mast Cell Biology.

Mast cells (MCs) are heterogeneous tissue-resident effector cells thought to play central roles in allergic inflammatory disease, yet the degree of heterogeneity and nature of these roles has remained elusive. In recent years, advances in tissue culture systems, pre-clinical mouse models, and the continued spread of single-cell RNA sequencing has greatly advanced our understanding of MC phenotypes in health and disease. These approaches have identified novel interactions of MC subsets with immune cells, neurons, and tissue structural cells, changing our understanding of how MCs both drive and help resolve tissue inflammation, reshape tissue microenvironments, and influence host behavior. This review addresses key studies from 2022-2024 that have advanced our understanding of MC biology in mouse and human.

Cardiovascular Disease and Cancer: A Dangerous Liaison.

The field of cardio-oncology has traditionally focused on the impact of cancer and its therapies on cardiovascular health. Mounting clinical and preclinical evidence, however, indicates that the reverse may also be true: cardiovascular disease can itself influence tumor growth and metastasis. Numerous epidemiological studies have reported that individuals with prevalent cardiovascular disease have an increased incidence of cancer. In parallel, studies using preclinical mouse models of myocardial infarction, heart failure, and cardiac remodeling support the notion that cardiovascular disorders accelerate the growth of solid tumors and metastases. These findings have ushered in a new and burgeoning field termed reverse cardio-oncology that investigates the impact of cardiovascular disease cause and pathophysiology on cancer emergence and progression. Recent studies have begun to illuminate the mechanisms driving this relationship, including shared risk factors, reprogramming of immune responses, changes in gene expression, and the release of cardiac factors that result in selective advantages for tumor cells or their local milieu, thus exacerbating cancer pathology. Here, we review the evidence supporting the relationship between cardiovascular disease and cancer, the mechanistic pathways enabling this connection, and the implications of these findings for patient care.

Immune-checkpoint-inhibitor therapy directed against PD-L1 is tolerated in the heart without manifestation of cardiac inflammation in a preclinical reversible melanoma mouse model.

Immune-checkpoint-inhibitors (ICI) target key regulators of the immune system expressed by cancer cells that mask those from recognition by the immune system. They have improved the outcome for patients with various cancer types, such as melanoma. ICI-based therapy is frequently accompanied by immune-related adverse side effects (IRAEs). The reversible melanoma cancer mouse model (B16F10 cells stably expressing a ganciclovir (GCV)-inducible suicide gene in C57BL/6N mice: B16F10-GCV) allows chemotherapy-free tumor elimination in advanced disease stage and demonstrates almost complete recovery of the mouse heart from cancer-induced atrophy, molecular impairment and heart failure. Thus, enabling the study of anti-cancer-therapy effects. Here, we analyzed potential cardiac side effects of antibody-mediated PD-L1 inhibition in the preclinical B16F10-GCV mouse model after tumor elimination and 2weeks recovery (50days after tumor inoculation). Anti-PD-L1 treatment was associated with improved survival as compared to isotype control (Ctrl) treated mice. Surviving anti-PD-L1 and Ctrl mice showed similar cardiac function, dimensions and the expression of cardiac stress and hypertrophy markers. Although anti-PD-L1 treatment was associated with increased troponin I type 3 cardiac (TNNI3) blood levels, cardiac mRNA expression of macrophage markers and elevated cardiac levels of secreted inflammatory factors compared to Ctrl treatment, both groups showed a comparable density of inflammatory cells in the heart (using CXCR4-ligand 68Ga-Pentixafor in PET-CT and immunohistochemistry). Thus, anti-PD-L1 therapy improved survival in mice with advanced melanoma cancer with no major cardiac phenotype or inflammation 50days after tumor inoculation. Without a second hit that triggers the inflammatory response, anti-PD-L1 treatment appears to be safe for the heart in the preclinical melanoma mouse model.

Hierarchically Engineered Self-Adaptive Nanoplatform Guided Intuitive and Precision Interventions for Deep-Seated Glioblastoma.

Glioblastoma multiforme (GBM), particularly the deep-seated tumor where surgical removal is not feasible, poses great challenges for clinical treatments due to complicated biological barriers and the risk of damaging healthy brain tissue. Here, we hierarchically engineer a self-adaptive nanoplatform (SAN) that overcomes delivery barriers by dynamically adjusting its structure, surface charge, particle size, and targeting moieties to precisely distinguish between tumor and parenchyma cells. We further devise a SAN-guided intuitive and precision intervention (SGIPi) strategy which obviates the need for sophisticated facilities, skilled operations, and real-time magnetic resonance imaging (MRI) guidance required by current MRI-guided laser or ultrasound interventions. In a preclinical intracranial GBM mouse model, SGIPi-based photodynamic therapy effectively impedes GBM progression with high tumor specificity and significantly extends overall survival. Moreover, the SGIPi potentiates chemotherapy while minimizing adverse effects; it eradicates intracranial GBM lesions in 100% cases solely through Temozolomide chemotherapy. This SGIPi strategy holds potential to improve the clinical management of GBM, with the possibility of extending survival rates and even achieving complete remission, and may inspire research focus from expensive and complex hardware development to simpler, delivery-based GBM interventions.

Development of Proton Density Fat Fraction Micro-MRI for Non-Invasive Quantitative Assessment of Bone Marrow Changes with Age and Radiation in Mouse Models.

Bone marrow (BM) adipocytes are critical in progressing solid tumor metastases and hematological malignancies across pediatric to aging populations. Single-point biopsies remain the gold standard for monitoring BM diseases, including hematologic malignancies, but are limited in capturing the full complexity of loco-regional and global BM microenvironments. Non-invasive imaging techniques like Magnetic Resonance Imaging (MRI), could offer valuable alternatives for real-time evaluation of BM diseases in both preclinical translational and clinical studies. We developed a preclinical proton density fat fraction (PDFF) MRI technique for quantitative BM composition assessment, focusing on fat fraction (FF) within mouse femurs. We validated this method using aging mice and young mice subjected to 10 Gy X-ray irradiation, compared with young unirradiated mice as controls. Water-fat phantoms (0% to 100% fat content) were used to optimize the imaging sequence, and immunohistochemical (IHC) staining with H&E validates equivalent adipose content in the femur BM regions. Significant differences in FF were observed across age groups (p = 0.001 for histology and p = 0.0002 for PDFF) and between irradiated and control mice (p = 0.005 for histology and p = 0.002 for PDFF). A strong correlation (R 2 ∼ 0.84) between FF values from PDFF and histology validates the accuracy of the technique. These findings demonstrate the potential of PDFF MRI as a non-invasive real-time imaging biomarker for quantifying BM fat fraction in preclinical mice model studies, particularly in evaluating the effects of aging, disease progression, and irradiation therapy in pediatric and translational oncology research.

AI Methods for Antimicrobial Peptides: Progress and Challenges.

Antimicrobial peptides (AMPs) are promising candidates to combat multidrug-resistant pathogens. However, the high cost of extensive wet-lab screening has made AI methods for identifying and designing AMPs increasingly important, with machine learning (ML) techniques playing a crucial role. AI approaches have recently revolutionised this field by accelerating the discovery of new peptides with anti-infective activity, particularly in preclinical mouse models. Initially, classical ML approaches dominated the field, but recently there has been a shift towards deep learning (DL) models. Despite significant contributions, existing reviews have not thoroughly explored the potential of large language models (LLMs), graph neural networks (GNNs) and structure-guided AMP discovery and design. This review aims to fill that gap by providing a comprehensive overview of the latest advancements, challenges and opportunities in using AI methods, with a particular emphasis on LLMs, GNNs and structure-guided design. We discuss the limitations of current approaches and highlight the most relevant topics to address in the coming years for AMP discovery and design.

Weight trajectories in aging humanized APOE mice with translational validity to human Alzheimer's risk population: A retrospective analysis.

Translational validity of mouse models of Alzheimer's disease (AD) is variable. Because change in weight is a well-documented precursor of AD, we investigated whether diversity of human AD risk weight phenotypes was evident in a longitudinally characterized cohort of 1,196 female and male humanized APOE (hAPOE) mice, monitored up to 28 months of age which is equivalent to 81 human years. Autoregressive Hidden Markov Model (AHMM) incorporating age, sex, and APOE genotype was employed to identify emergent weight trajectories and phenotypes. In the hAPOE-AD mouse cohort, five distinct weight trajectories emerged: three trajectories were associated with a weight loss phenotype (36% of mice, n = 426), one with weight gain (13% of mice, n = 152), and one trajectory of no change in weight (34% of mice, n = 403). The AHMM model findings were validated with post-hoc survival analyses, revealing differences in survival rates across the five identified phenotypes. Further validation was performed using body composition and plasma β-amyloid data from mice within the identified gain, loss and stable weight trajectories. Weight gain trajectory was associated with elevated plasma β-amyloid levels, higher body fat composition, lower survival rates and a greater proportion of APOE4/4 carriers. In contrast, weight loss was associated with greater proportion of hAPOE3/4 carriers, better survival rates and was predominantly male. The association between weight change and AD risk observed in humans was mirrored in the hAPOE-AD mouse model. Weight trajectories of APOE3/3 mice were equally distributed across weight gain, loss and stability. Surprisingly, despite genetic uniformity, comparable housing, diet and handling, distinct weight trajectories and divergence points emerged for subpopulations. These data are consistent with the heterogeneity observed in the human population for change in body weight during aging and highlight the importance of longitudinal phenotypic characterization of mouse aging to advance the translational validity of preclinical AD mouse models.

Temporally and Spatially Controlled Age-Related Prostate Cancer Model in Mice.

The initiation and progression of prostate cancer (PCa) are associated with aging. In the history of age-related PCa research, mice have become a more popular animal model option than any other species due to their short lifespan and rapid reproduction. However, PCa in mice is usually induced at a relatively young age, while it spontaneously develops in humans at an older age. Thus, it is essential to develop a method by which the PCa initiation and progression timeline can be strictly controlled to mimic human physiological conditions. One milestone in this field was the identification of the prostate-specific transcription factor, Probasin (Pb), which allowed for the prostate-specific expression of genes knocked into the mice's genome. Another milestone is the establishment of the preclinical mouse model with Pten conditionally knocked out in the prostate tissue, which closely mimics the formation and growth of human PCa. Hereby, we present the prostate-specific temporally and spatially controlled Pten knockout PCa mouse model that can be induced using an adenovirus-based Cre-LoxP system. The Cre recombinase (Cre) is inserted into an adenovirus vector. Unlike Pb-Cre knock-in models (which are spatially but not temporally controlled), the expression of Cre is activated to knock out Pten from the mice's prostate epithelial cells once injected. The viral delivery procedures strictly control the location and time of Pten knockout. This novel approach provides a powerful age-related murine model for PCa, emphasizing the effect of aging on prostate carcinogenesis. Key features • In vivo delivery of Cre recombinase adenovirus (Ad-Cre-Luc) in Pten LoxP/LoxP (L/L) mice. • Generation of Cre-expressing Ad-Cre-Luc-mediated ablation of Pten in anterior prostate epithelial cells of adult Pten L/L mice at different ages. • The Ad-Cre-Luc-mediated ablation of Pten leads to hyperplasia that progresses through prostatic intraepithelial neoplasia (PIN) to adenocarcinoma. • PIN refers to the non-cancerous growth of epithelial cells in the prostate tissue-not cancer but a precursor of prostate cancer [1].

TARGETING BIOLOGICAL AGING MECHANISMS OF LOWER URINARY TRACT SYMPTOMS IN OLDER MEN WITH AN EXERCISE INTERVENTION

Abstract Physical frailty is associated with lower urinary tract symptoms (LUTS) severity and progression among older men with suspected benign prostatic hyperplasia (BPH). Prospective cohort studies have consistently demonstrated that both physically inactive and frail men are more likely to develop BPH or experience LUTS progression. The combination of aerobic plus resistance exercise has been demonstrated in clinical trials to have pleotropic effects that delay, prevent, or treat many frailty-related conditions that often co-occur with LUTS and BPH, including cardiovascular disease, diabetes, and erectile dysfunction. Although it has never been tested in humans, pre-clinical mouse models of BPH demonstrate improved voiding behaviors after exposure to exercise and exercise is known to improve several mechanisms that are implicated in human LUTS/BPH pathophysiology, including insulin resistance, endocrine dysregulation, and mitochondrial dysfunction. The recently launched PROUD study (NCT06225479; MPIs: Bauer, Kenfield) is a 2-arm pilot randomized controlled trial among physically inactive older men with LUTS/BPH testing the effect of a 12-week remote exercise intervention versus health education control. Outcomes include: 1) feasibility, acceptability, fidelity, and safety; 2) changes in LUTS, urologic and physical function, and physical activity; and 3) feasibility of detecting exercise-induced change in novel frailty-related mechanistic measures. This symposium session will review the scientific rationale, key study design and analytic decisions, enrollment progress thus far, and collaborative opportunities using stored biobank specimens.

Escalation of intravenous fentanyl self-administration and assessment of withdrawal behavior in male and female mice.

The rise in overdose deaths from synthetic opioids, especially fentanyl, necessitates the development of preclinical models to study fentanyl use disorder (FUD). While there has been progress with rodent models, additional translationally relevant models are needed to examine excessive fentanyl intake and withdrawal signs. The current study aimed to develop a translationally relevant preclinical mouse model of FUD by employing chronic intravenous fentanyl self-administration (IVSA). The study performed intravenous self-administration (IVSA) of fentanyl in male and female C57BL/6J mice for 14 days. Mechanical pain sensitivity during withdrawal was assessed using the von Frey test. Anxiety-like behavior was evaluated via the open field test one week into abstinence, and drug seeking behavior after extended abstinence was assessed at four weeks abstinence. Both male and female mice demonstrated a significant escalation in fentanyl intake over the 14 days of self-administration, with significant front-loading observed in the final days of self-administration. Mice showed increased mechanical pain sensitivity at 36 and 48hours withdrawal from fentanyl. At 1-week abstinence from fentanyl, mice exhibited increased anxiety-like behavior compared to naive mice. Four weeks into abstinence from fentanyl, mice maintained lever-pressing behavior on the previous reward-associated active lever, with significantly higher active lever pressing compared to inactive lever pressing. The study establishes a translationally relevant mouse model of IVSA of fentanyl, effectively encapsulating critical aspects of FUD, including escalation of drug intake, front-loading behavior, withdrawal signs, and drug-seeking behavior into extended abstinence. This model offers a robust basis for further exploration into behavioral and neurobiological mechanisms involved in fentanyl dependence and potential therapeutic strategies.

Matrix-free human lung organoids derived from induced pluripotent stem cells to model lung injury

BackgroundOrganoids, as near-physiological 3D culture systems, offer new opportunities to study the pathogenesis of various organs in mimicking the cellular complexity and functionality of human organs.MethodHere we used a quite simple and very practicable method to successfully generate induced pluripotent stem cell (iPSC)-derived human lung organoids (LuOrg) in a matrix-free manner as an alternative to the widely used preclinical mouse models in order to investigate normal lung damage in detail and as close as possible to the patient. We performed detailed morphological and molecular analyses, including bulk and single cell RNA sequencing, of generated lung organoids and evaluated the quality and robustness of our model as a potential in vitro platform for lung diseases, namely radiation-induced lung injury.ResultsA matrix-free method for differentiation of iPSCs can be used to obtain lung organoids that morphologically reflect the target tissue of the human lung very well, especially with regard to the cellular composition. The different cellular fates were investigated following the genotoxic stress induced by radiation and revealed further insights in the radiation-sensitivity of the different lung cells. Finally, we provide cellular gene sets found to be induced in the different lung organoid cellular subsets after irradiation, which could be used as additional RT response and particularly senescence gene sets in future studies.ConclusionBy establishing these free-floating LuOrgs for the investigation of cancer therapeutic approaches as a new and patient-oriented in vitro platform particularly in experimental radiooncology, not only a reduction in the number of experimental animals, but also an adequately and meaningfully replacement of corresponding animal experiments can be achieved.

Engineered extrachromosomal oncogene amplifications promote tumorigenesis

Focal gene amplifications are among the most common cancer-associated mutations1 but have proven challenging to engineer in primary cells and model organisms. Here we describe a general strategy to engineer large (more than 1 Mbp) focal amplifications mediated by extrachromosomal DNAs (ecDNAs)2 in a spatiotemporally controlled manner in cells and in mice. By coupling ecDNA formation with expression of selectable markers, we track the dynamics of ecDNA-containing cells under physiological conditions and in the presence of specific selective pressures. We also apply this approach to generate mice harbouring Cre-inducible Myc- and Mdm2-containing ecDNAs analogous to those occurring in human cancers. We show that the engineered ecDNAs spontaneously accumulate in primary cells derived from these animals, promoting their proliferation, immortalization and transformation. Finally, we demonstrate the ability of Mdm2-containing ecDNAs to promote tumour formation in an autochthonous mouse model of hepatocellular carcinoma. These findings offer insights into the role of ecDNA-mediated gene amplifications in tumorigenesis. We anticipate that this approach will be valuable for investigating further unresolved aspects of ecDNA biology and for developing new preclinical immunocompetent mouse models of human cancers harbouring specific focal gene amplifications.

Investigation of the synergic effect of mocetinostat and capecitabine in a triple-negative breast neoplasms mouse model.

Combined administration of two or more drugs is emerging as a new strategy in triple-negative breast neoplasms. This is the first study to investigate the combination of the histone deacetylase inhibitor mocetinostat and the antimetabolite drug capecitabine in triple-negative mammary neoplasms in a preclinical mouse model. Thirty-five female mice were grouped into the control group, capecitabine group, mocetinostat group, and combined drugs group. At the end of the experimental period, body weight, and tumor weight were measured and tumor tissue and lung tissue were histologically examined. The results showed that the body weight of mice in the drug-treated groups was reduced by about 18%. Tumor weights were also reduced by 21% in the mocetinostat group, 27.5% in the capecitabine group, and 45% in the combined group. The combination of mocetinostat and capecitabine decreased the formation of tumors and metastases in lung tissue. In summary, the combination of mocetinostat and capecitabine was more effective than either drug alone in reducing the size of triple-negative breast neoplasms in a mouse model.