Advances In Preclinical Research Research Articles

IMMU-22. B7-H3 NANOBODY CAR-T AND SOLUBLE TREM2 GENETICALLY ENGINEERED MYELOID CELL THERAPY IN MURINE SYNGENEIC GLIOBLASTOMA

Abstract BACKGROUND Myeloid immunosuppression can be a major impediment to CAR-T cell therapy for CNS tumors. Importantly, CAR-T cells are often evaluated in immunocompromised mice which cannot accurately recapitulate the tumor immune response or tumor microenvironment. CNS tumor macrophages express TREM2, an important driver of immunosuppression. This study combines B7-H3 targeting nanobody CAR-T cells which are cross reactive in humans and mice (currently being evaluated for clinical development) with genetically engineered myeloid cells (GEMys) expressing soluble TREM2 (sTREM2) in a syngeneic mouse model of glioblastoma (GBM), with the aim of blocking TREM2-mediated immunosuppression to enhance CAR-T cell function. METHODS Syngeneic murine GBM, designed to mimic human GBM and generated at the Center for Advanced Preclinical Research (CAPR), was used for all studies. B7-H3 nanobody CAR-T cells were generated from mouse splenic T cells. In vitro tumor killing assays were performed by xCELLigence. sTREM2 GEMys were generated from bone marrow progenitor cells using lentiviral transduction. For in vivo studies, mice underwent orthotopic implantation of tumors and placement of an MRI-compatible, permanent catheter-port system for serial intracerebroventricular (ICV) administration of cell therapies. Mice were treated with CAR-T and/or GEMy therapies, evaluated weekly for tumor burden by MRI, and were monitored for survival. RESULTS Syngeneic GBM expression of B7-H3 was confirmed by flow cytometry, immunofluorescence, and western blot. B7-H3 nanobody CAR-T cells demonstrated effective killing of GBM by xCELLigence. sTREM2 GEMys were successfully generated and confirmed to produce sTREM2. Cell therapies were successfully infused ICV via the implanted catheter-port system, demonstrating safety and feasibility of this model for serial ICV cellular therapy. CONCLUSIONS B7-H3 nanobody CAR-T cells were validated against syngeneic GBM, and we have demonstrated safety and feasibility of ICV catheter administration of cellular therapies in vivo. Future studies will continue to evaluate in vivo timing and efficacy of CAR-T and GEMy therapy.

AAV-Mediated Restoration of Dystrophin-Dp71 in the Brain of Dp71-Null Mice: Molecular, Cellular and Behavioral Outcomes.

A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the DMD gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood-brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients.

Pain Research and Treatment: Policy Implications

Pain is a subjective experience for which we lack a thorough mechanistic understanding. While it has often been considered a secondary symptom of a disease, research has revealed that chronic pain can be recognized as a disease of its own. Preclinical studies often use animal models to understanding the pathology and test potential therapies with clinical trials of novel analgesics often showing underwhelming results. From a clinician's perspective, pain can be challenging to treat without incurring undesirable drug effects. Pain research, diagnosis, and treatment have changed dramatically over the last two decades. This has coincided with shifts in reliance on opioid-based approaches to managing pain. Here we present an overview of recent advances in preclinical research, novel approaches to diagnosis and treatment, along with considerations for animal care. Collectively, this report may provide a guide on which to base relevant policy changes that would meet the demands of societal need and satisfy the need for scientific evidence.

Mitochondria-Targeted Antioxidant Therapeutics for Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major global health problem that affects both civilian and military populations worldwide. Post-injury acute, sub-acute, and chronic progression of secondary injury processes may contribute further to other neurodegenerative diseases. However, there are no approved therapeutic options available that can attenuate TBI-related progressive pathophysiology. Recent advances in preclinical research have identified that mitochondria-centric redox imbalance, bioenergetics failure and calcium dysregulation play a crucial role in secondary injury progression after TBI. Mitochondrial antioxidants play an important role in regulating redox homeostasis. Based on the proven efficacy of preclinical and clinical compounds and targeting numerous pathways to trigger innate antioxidant defense, we may be able to alleviate TBI pathology progression by primarily focusing on preserving post-injury mitochondrial and cerebral function. In this review, we will discuss novel mitochondria-targeted antioxidant compounds, which offer a high capability of successful clinical translation for TBI management in the near future.

Lymphedema-associated fibroblasts are a TGF-β1 activated myofibroblast subpopulation related to fibrosis and stage progression in patients and a murine microsurgical model.

The driver of secondary lymphedema (SL) progression is chronic inflammation, which promotes fibrosis. Despite advances in preclinical research, a specific effector cell subpopulation as a biomarker for therapy response or stage progression is still missing for SL. Whole skin samples of 35 murine subjects of a microsurgical-induced SL model and 12 patients with SL were collected and their fibroblasts were isolated. These lymphedema-derived fibroblasts (LAF) were cultured in a collagen I-poly-D-Lysine 3D hydrogel to mimic skin conditions. Fibroblasts from non-lymphedema skin were used as negative control and TGF-β-stimulated fibroblasts were used to recreate profibrotic myofibroblasts. Quantitative immunocytofluorescence confocal microscopy analysis and invasion functional assays were performed in all subpopulations and statistically compared. In contrast to normal skin fibroblasts, LAF exhibit α-SMA-positive stress fibers and a reduced number of tight junctions in 3D hydrogel conditions. The switch from normal E-cadherin high phenotype to an N-cadherin high-E-cadherin low morphology suggests epithelial to mesenchymal transition for expansion and proliferation. This pathological behavior of LAF was confirmed by live cell imaging analysis of invasion assays. The significant activation of markers of the TGFBR2-Smad pathway and collagen synthesis (HSP-47) in LAF supports EMT phenotypic changes and previous findings relating to TGF-β1 and fibrosis with lymphedema. A characteristic SL myofibroblast subpopulation was identified and translationally related to fibrosis and TGF-β1-associated stage progression. This SL-related subpopulation was termed lymphedema-associated fibroblasts. A comprehensive stage-related characterization is required to validate LAF as a reliable biomarker for SL disease progression.

Immunomodulatory Mechanism and Potential Application of Dental Pulp-Derived Stem Cells in Immune-Mediated Diseases.

Dental pulp stem cells (DPSCs) are mesenchymal stem cells (MSCs) derived from dental pulp tissue, which have high self-renewal ability and multi-lineage differentiation potential. With the discovery of the immunoregulatory ability of stem cells, DPSCs have attracted much attention because they have similar or even better immunomodulatory effects than MSCs from other sources. DPSCs and their exosomes can exert an immunomodulatory ability by acting on target immune cells to regulate cytokines. DPSCs can also migrate to the lesion site to differentiate into target cells to repair the injured tissue, and play an important role in tissue regeneration. The aim of this review is to summarize the molecular mechanism and target cells of the immunomodulatory effects of DPSCs, and the latest advances in preclinical research in the treatment of various immune-mediated diseases, providing new reflections for their clinical application. DPSCs may be a promising source of stem cells for the treatment of immune-mediated diseases.

Advances and Challenges of Cannabidiol as an Anti-Seizure Strategy: Preclinical Evidence.

The use of Cannabis for medicinal purposes has been documented since ancient times, where one of its principal cannabinoids extracted from Cannabis sativa, cannabidiol (CBD), has emerged over the last few years as a promising molecule with anti-seizure potential. Here, we present an overview of recent literature pointing out CBD's pharmacological profile (solubility, metabolism, drug-drug interactions, etc.,), CBD's interactions with multiple molecular targets as well as advances in preclinical research concerning its anti-seizure effect on both acute seizure models and chronic models of epilepsy. We also highlight the recent attention that has been given to other natural cannabinoids and to synthetic derivatives of CBD as possible compounds with therapeutic anti-seizure potential. All the scientific research reviewed here encourages to continue to investigate the probable therapeutic efficacy of CBD and its related compounds not only in epilepsy but also and specially in drug-resistant epilepsy, since there is a dire need for new and effective drugs to treat this disease.

Novel approaches for periodontal tissue engineering.

The periodontal complex involves the hard and soft tissues which support dentition, comprised of cementum, bone, and the periodontal ligament (PDL). Periodontitis, a prevalent infectious disease of the periodontium, threatens the integrity of these tissues and causes irreversible damage. Periodontal therapy aims to repair and ultimately regenerate these tissues toward preserving native dentition and improving the physiologic integration of dental implants. The PDL contains multipotent stem cells, which have a robust capacity to differentiate into various types of cells to form the PDL, cementum, and alveolar bone. Selection of appropriate growth factors and biomaterial matrices to facilitate periodontal regeneration are critical to recapitulate the physiologic organization and function of the periodontal complex. Herein, we discuss the current state of clinical periodontal regeneration including a review of FDA-approved growth factors. We will highlight advances in preclinical research toward identifying additional growth factors capable of robust repair and biomaterial matrices to augment regeneration similarly and synergistically, ultimately improving periodontal regeneration's predictability and long-term efficacy. This review should improve the readers' understanding of the molecular and cellular processes involving periodontal regeneration essential for designing comprehensive therapeutic approaches.

Electrospun extracellular matrix scaffold improves cardiac structure and function post‐myocardial infarction

AbstractThe current unmet clinical need for post‐myocardial infarction (MI) treatments has driven the development of diverse scaffolds for regenerating the infarcted area, based on natural and synthetic polymers. Decellularized porcine cardiac extracellular matrix (pcECM) has emerged as a promising biomaterial for cardiac regeneration, due to its unique bioactivity and microstructure that mimic the natural tissue. We have previously reported the development of an electrospun pcECM cardiac scaffold that was shown to preserve the mechanical, structural, and biological properties of cardiac ECM while allowing a controllable reproducible production. In the present work, however, we reveal the potential of this unique scaffold as a possible treatment post‐MI. Two types of electrospun pcECM scaffolds, varying in thickness, were applied to rat hearts, 4 weeks following MI induction, thus allowing cardiac deterioration and a scar tissue formation before treatment (chronic model). Our results show moderated remodeling, decreased scar, and reduced wall thinning accompanied by a partial functional recovery in the hearts of both treatment groups when compared to the control. When examining the different structural and functional parameters of the heart, however, the advantage of the thicker electrospun pcECM scaffold is revealed. Hence, significant improvement was obtained in important structural parameters such as wall thickness and functional parameters such as ejection fraction. Altogether, our results indicate the potential applicability of electrospun pcECM scaffolds for cardiac regeneration, establishing the basis for advanced preclinical research using large animal models.

Neurogenic Inflammation: The Participant in Migraine and Recent Advancements in Translational Research.

Migraine is a primary headache disorder characterized by a unilateral, throbbing, pulsing headache, which lasts for hours to days, and the pain can interfere with daily activities. It exhibits various symptoms, such as nausea, vomiting, sensitivity to light, sound, and odors, and physical activity consistently contributes to worsening pain. Despite the intensive research, little is still known about the pathomechanism of migraine. It is widely accepted that migraine involves activation and sensitization of the trigeminovascular system. It leads to the release of several pro-inflammatory neuropeptides and neurotransmitters and causes a cascade of inflammatory tissue responses, including vasodilation, plasma extravasation secondary to capillary leakage, edema, and mast cell degranulation. Convincing evidence obtained in rodent models suggests that neurogenic inflammation is assumed to contribute to the development of a migraine attack. Chemical stimulation of the dura mater triggers activation and sensitization of the trigeminal system and causes numerous molecular and behavioral changes; therefore, this is a relevant animal model of acute migraine. This narrative review discusses the emerging evidence supporting the involvement of neurogenic inflammation and neuropeptides in the pathophysiology of migraine, presenting the most recent advances in preclinical research and the novel therapeutic approaches to the disease.

The Value of Mouse Models of Rare Diseases: A Spanish Experience.

Animal models are invaluable for biomedical research, especially in the context of rare diseases, which have a very low prevalence and are often complex. Concretely mouse models provide key information on rare disease mechanisms and therapeutic strategies that cannot be obtained by using only alternative methods, and greatly contribute to accelerate the development of new therapeutic options for rare diseases. Despite this, the use of experimental animals remains controversial. The combination of respectful management, ethical laws and transparency regarding animal experimentation contributes to improve society’s opinion about biomedical research and positively impacts on research quality, which eventually also benefits patients. Here we present examples of current advances in preclinical research in rare diseases using mouse models, together with our perspective on future directions and challenges.

Insight into epigenetics of human endometriosis organoids: DNA methylation analysis of HOX genes and their cofactors

To evaluate and compare the methylation pattern of Human Homeobox (HOX) clusters (A-D) and HOX cofactors in normal, eutopic, and ectopic endometrial tissues with ectopic and eutopic endometriosis organoids as advanced preclinical research models. A chromatin immunoprecipitation (ChIP) array containing 84 genes was used to analyze methylation levels of HOX clusters (A-D) and HOX cofactors in normal, eutopic, and ectopic endometrial biopsy specimens as well as ectopic and eutopic endometriosis organoids. Reproductive biomedicine and cell science research centers. Nine healthy women without endometriosis (control) and 16 women diagnosed with endometriosis. Ectopic endometrial lesions were obtained using a laparoscopic procedure, and eutopic and control endometrium biopsy specimens were obtained using pipelle sampling. Methylation levels of HOX clusters (A-D) and HOX cofactors in eutopic and ectopic endometrial biopsy specimens, as well as eutopic and ectopic endometriosis organoids and normal endometrium. Most HOX clusters (A-D) and HOX cofactors showed methylation alterations in ectopic/eutopic endometrial tissues and ectopic/eutopic endometriosis organoids compared with normal endometrium. These methylation alterations had the same pattern in ectopic/eutopic tissue biopsy specimens and ectopic/eutopic endometriosis organoids in most genes. A contrariwise methylation pattern was observed in 28 of 84 genes in the ectopic/eutopic tissue biopsy specimens and ectopic/eutopic endometriosis organoids. Because a conserved pattern of methylation alterations in endometriosis tissues and organoids was observed for most of the investigated genes (56 of 84), it can be concluded that endometriosis organoids maintain epigenetic changes. Therefore, our study suggests endometriosis organoids as a novel preclinical model to determine the epigenetic mechanisms that underlie endometriosis.

Mesenchymal Stem/Stromal Cell-Mediated Mitochondrial Transfer and the Therapeutic Potential in Treatment of Neurological Diseases.

Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells that can be derived from various tissues. Due to their regenerative and immunomodulatory properties, MSCs have been extensively researched and tested for treatment of different diseases/indications. One mechanism that MSCs exert functions is through the transfer of mitochondria, a key player involved in many biological processes in health and disease. Mitochondria transfer is bidirectional and has an impact on both donor and recipient cells. In this review, we discussed how MSC-mediated mitochondrial transfer may affect cellular metabolism, survival, proliferation, and differentiation; how this process influences inflammatory processes; and what is the molecular machinery that mediates mitochondrial transfer. In the end, we summarized recent advances in preclinical research and clinical trials for the treatment of stroke and spinal cord injury, through application of MSCs and/or MSC-derived mitochondria.

Recent Developments of Novel Pharmacologic Therapeutics for Prevention of Chemotherapy-Induced Peripheral Neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and dose-limiting toxicity, negatively affecting both quality of life and disease outcomes. To date, there is no proven preventative strategy for CIPN. Although multiple randomized trials have evaluated a variety of pharmacologic interventions for the treatment of CIPN, only duloxetine has shown clear efficacy in a phase III study. The National Cancer Institute's Symptom Management and Health-Related Quality of Life Steering Committee has identified CIPN as a priority for translational research in cancer care. Promising advances in preclinical research have identified several novel preventative and therapeutic targets, which have the potential to transform the care of patients with this debilitating neurotoxicity. Here, we provide an overarching view of emerging strategies and therapeutic targets that are currently being evaluated in CIPN.

Depicting brighter possibilities for treating blindness.

Advances in preclinical research are now being translated into innovative clinical solutions for blindness.

Developing Repetitive Transcranial Magnetic Stimulation (rTMS) as a Treatment Tool for Cocaine Use Disorder: a Series of Six Translational Studies.

Cocaine dependence is a chronic and relapsing disorder which is particularly resistant to behavioral or pharmacologic treatment, and likely involves multiple dysfunctional frontal-striatal circuits. Through advances in preclinical research in the last decade, we now have an unprecedented understanding of the neural control of drug-taking behavior. In both rodent models and human clinical neuroimaging studies, it is apparent that medial frontal-striatal limbic circuits regulate drug cue-triggered behavior. While non-human preclinical studies can use invasive stimulation techniques to inhibit drug cue-evoked behavior, in human clinical neuroscience, we are pursuing non-invasive theta burst stimulation (TBS) as a novel therapeutic tool to inhibit drug cue-associated behavior. Our laboratory and others have spent the last 7 years systematically and empirically developing a non-invasive, neural circuit-based intervention for cocaine use disorder. Utilizing a multimodal approach of functional brain imaging and brain stimulation, we have attempted to design and optimize a repetitive transcranial magnetic stimulation treatment protocol for cocaine use disorder. This manuscript will briefly review the data largely from our own lab that motivated our selection of candidate neural circuits, and then summarize the results of six studies, culminating in the first double-blinded, sham-controlled clinical trial of TMS as a treatment adjuvant for treatment-engaged cocaine users (10 sessions, medial prefrontal cortex, 110% resting motor threshold, continuous theta burst stimulation, 3600 pulses/session). The intent of this review is to highlight one example of a systematic path for TMS treatment development in patients. This path is not necessarily optimal, exclusive, or appropriate for every neurologic or psychiatric disease. Rather, it is one example of a reasoned, empirically derived pathway which we hope will serve as scaffolding for future investigators seeking to develop TMS treatment protocols.

Abstract 1479: Preclinical model of human melanoma for evaluation of targeted drug treatment and for immunotherapy validation

Abstract Malignant melanoma accounts for less than 5% of skin cancer cases, yet it represents 75% of deaths from skin cancer. The high mortality rate is due to the malignant, metastatic nature of the disease and resistance to chemotherapeutic treatments. Most mouse melanoma models have not fully recapitulated the histopathology of the disease and its metastatic nature. At NCI's Center for Advanced Preclinical Research (CAPR), we have adapted the HGF/SF; CDK4R24C transgenic mouse model to an optimized allograft transplant model for preclinical therapeutic studies in primary and metastatic melanoma. This genetically engineered mouse-derived Allograft (GDA) model recapitulates the features of the original GEM, including the epithelioid histopathology and key marker expression of human melanoma. It is an efficient and tractable tool for monitoring of both tumor growth and therapeutic responses in primary and metastatic melanoma in the context of a normal immune system. Additionally, aberrant expression of c-Met and upregulation of the downstream signaling pathway in HGF-GDA tumors is relevant for targeted therapeutics in melanoma. Thus, the model is a useful platform for evaluating therapies that target tumor cells and/or immunomodulatory pathways in intervention or adjuvant settings. Although drugs such as the c-Met inhibitor crizotinib and the MEK inhibitor trametinib were potent in cell culture, PD analyses of short-term (4-6 hour) treatment with small molecule therapies indicated that treatment incompletely suppresses the pathway in vivo compared to the corresponding primary cell line, and does not inhibit tumor growth. Therefore the HGF-GDA can be exploited to examine combination treatments that either prevent feedback activation of downstream pathway nodes in vivo, or modify alternate pathways, such as immunomodulatory targets. Hence, we are currently exploring rational combinations of oncogene-targeted therapy with immune-targeted therapy, for example, combined trametinib and anti-CTLA4 antibody treatment. In the HGF-GDA, complete response was observed in a subgroup of mice treated with anti-CTLA-4, i.e. established tumors fully regressed, yet the durable response and increased survival time (based on tumor volume) was not enhanced by concurrent treatment with trametinib. Future treatment studies will involve alternative regimens. Additionally, since metastasis, not the primary tumor, leads to progression of melanoma in patients, we have characterized a primary tumor resection model in which only metastatic disease is treated. Lung metastases develop after resection of the HGF-GDA primary tumor, which may be treated in an intervention or adjuvant setting. Therefore we evaluated the effect on survival of the same combination therapy (trametinib and anti-CTLA-4) we previously used to treat the primary tumor, but as adjuvant treatment for the metastatic melanoma model. Citation Format: Rajaa El Meskini, Michelle Gumprecht, Alan Kulaga, Anthony Iacovelli, Terry Van Dyke, Chi-Ping Day, Glenn Merlino, Zoe Weaver Ohler. Preclinical model of human melanoma for evaluation of targeted drug treatment and for immunotherapy validation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1479.

ChemInform Abstract: Spirooxindoles: Promising Scaffolds for Anticancer Agents

AbstractReview: 84 refs.

Computer-aided pulmonary image analysis in small animal models.

To develop an automated pulmonary image analysis framework for infectious lung diseases in small animal models. The authors describe a novel pathological lung and airway segmentation method for small animals. The proposed framework includes identification of abnormal imaging patterns pertaining to infectious lung diseases. First, the authors' system estimates an expected lung volume by utilizing a regression function between total lung capacity and approximated rib cage volume. A significant difference between the expected lung volume and the initial lung segmentation indicates the presence of severe pathology, and invokes a machine learning based abnormal imaging pattern detection system next. The final stage of the proposed framework is the automatic extraction of airway tree for which new affinity relationships within the fuzzy connectedness image segmentation framework are proposed by combining Hessian and gray-scale morphological reconstruction filters. 133 CT scans were collected from four different studies encompassing a wide spectrum of pulmonary abnormalities pertaining to two commonly used small animal models (ferret and rabbit). Sensitivity and specificity were greater than 90% for pathological lung segmentation (average dice similarity coefficient > 0.9). While qualitative visual assessments of airway tree extraction were performed by the participating expert radiologists, for quantitative evaluation the authors validated the proposed airway extraction method by using publicly available EXACT'09 data set. The authors developed a comprehensive computer-aided pulmonary image analysis framework for preclinical research applications. The proposed framework consists of automatic pathological lung segmentation and accurate airway tree extraction. The framework has high sensitivity and specificity; therefore, it can contribute advances in preclinical research in pulmonary diseases.

Novel Pancreatic Cancer Cell Lines Derived from Genetically Engineered Mouse Models of Spontaneous Pancreatic Adenocarcinoma: Applications in Diagnosis and Therapy

Pancreatic cancer (PC) remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM) models that produce spontaneous pancreatic adenocarcinoma (PDAC) have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC) mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a KrasG12D;Pdx1-Cre (KC) mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of KrasG12D;Trp53R172H;Pdx1-Cre (KPC) mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines (i.e. KrasG12D mutation was observed in all three cell lines while Trp53 mutation was observed only in KPC cell lines). The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic). The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.