Relevant Preclinical Models Research Articles

Development of a Clinically Relevant Preclinical Animal Model to Mimic Suburethral Implantation of Support Materials for Stress Urinary Incontinence.

Following the severe complications widely reported in some women following polypropylene suburethral mesh implantation for the treatment of stress urinary incontinence, it has become clear that careful preclinical study in an appropriate animal model is essential. The preclinical model described here allows the postoperative clinical and histochemical assessment of material implanted in the sheep vagina and represents a model that allows prediction of the potential vaginal tissue responses to suburethral mesh implantation before implantation into humans. To develop and evaluate a relevant preclinical animal model to mimic suburethral implantation of support materials for stress urinary incontinence. Fourteen parous ewes of 30-40 kg were used. Under general anesthetic, animals were placed in the lithotomy position. A 12-French Foley catheter was inserted. The ventral vaginal wall was longitudinally incised. The space between the vaginal wall and the urethra was developed, with lateral retropubic dissection. Either a standard transvaginal polypropylene tape (seven animals) or a fascia mimetic microfibre spun polyurethane tape (seven animals) were inserted using an introducer either via a suprapubic (seven animals) or a transvaginal approach (seven animals) into the suburethral and retropubic space. The incisions were closed, and the catheter was removed. A betadine vaginal pack was placed and removed after 24 h. Postoperatively, the animals were observed carefully for pain and complications and allowed access to water and food under continuous veterinary supervision adhering to USA-approved protocols. On the second postoperative day, all the animals passed urine freely and had regular bowel motions. No procedure related complications were observed. This is the first large animal model which has been designed for suburethral implantation. and which can be effectively used for the preoperative evaluation of novel materials designed for suburethral implantation. We believe that this technique in this large animal model accurately reproduces the surgical technique used to treat stress urinary incontinence in women. It provides a new and effective in vivo model for the objective evaluation of new materials to treat female stress incontinence.

Metabolic Syndrome Nonalcoholic Steatohepatitis Male Mouse With Adeno-Associated Viral Renin as a Novel Model for Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) currently accounts for more than half of patients with HF, with limited approved evidence-based therapies. HFpEF is a complex multifactorial disease associated with hypertension, obesity, diabetes, and renal dysfunction. In addition to our limited understanding of HFpEF pathophysiology, the development of new therapies is partially hindered by the existing translationally relevant preclinical HFpEF models. Here, we report the development of a novel 2-hit HFpEF male mouse model through adding hypertensive stress from the activation of the renin-angiotensin-aldosterone system with adeno-associated viral-renin (AAV-renin), to the metabolic syndrome nonalcoholic steatohepatitis (MSNASH, formerly FATZO) mouse (MSNASH+AAV-renin). To further demonstrate model translatability, MSNASH+AAV-renin mice were then treated with approved HFpEF therapies sacubitril/valsartan or a combination of sacubitril/valsartan and empagliflozin. We found that the MSNASH+AAV-renin mouse model demonstrates clinically relevant features of human HFpEF, including preserved ejection fraction, cardiac hypertrophy, left atrial enlargement, diastolic dysfunction, elevated natriuretic peptides, inflammation, and low exercise capacity. We also demonstrate that treatment with approved HFpEF therapies sacubitril/valsartan and the combination of sacubitril/valsartan with empagliflozin improves the cardiac phenotypes of HFpEF in this model. The MSNASH+AAV-renin model faithfully represents a severe cardiometabolic HFpEF phenogroup that can be used as a new tool for HFpEF preclinical research and drug discovery.

Abstract A002: Dissecting the brain metastatic microenvironment to uncover immune and molecular correlates of response to immunotherapy

Abstract Despite promising results with immune checkpoint blockade (ICB) therapy, brain metastases (BrM) remain a deadly complication for cancer patients. Understanding the highly specialized brain metastatic tumor microenvironment (BrTME) is crucial for identifying determinants of response, however, progress has been hindered by limited access to patient samples and scarcity of relevant preclinical models. Here, we developed two mouse melanoma BrM models by intracardiac injection of cells derived from our previously established M4-B2905 melanoma mouse cell line. We characterized their mutational landscape and performed single-cell phenotypic and transcriptomic analysis, demonstrating that these models recapitulate the cellular and molecular features of human melanoma BrMs. Comparative and interactome analysis of our models revealed key factors contributing to ICB response and resistance. We found that the responsive model (BR1) was characterized by tumor cells that polarize microglia toward reactive states via inflammatory programs and response to IFN signaling. These microglia express high levels of antigen presentation and immunostimulatory molecules, triggering T cell recruitment and activation and promoting ICB therapy response. In contrast, in the resistant model (BR3), the tumor cells express neurological molecular signatures and ligands that sustain microglia homeostasis. This results in poor T cell infiltration predominantly composed of naïve cells, evading immune reaction and promoting ICB resistance. While we showed that systemic ICB therapy induced BrTME changes in both models, differences between responder and resistant models were maintained, emphasizing the importance of tumor cell-intrinsic programs in dictating the BrTME. We validated the translational relevance of our findings and demonstrated that BR1 signatures positively correlate with T cell infiltration and are associated with improved patient outcomes, whereas BR3 signatures negatively correlate with T cells and are found in patients with worse prognosis. Here we address an important challenge in the field by providing clinically relevant BrM models that recapitulate both the cellular and molecular features of human disease and the variability of ICB response seen in patients. We further reveal mechanistic insights into BrM ICB response and identify potential therapeutic targets to modulate the BrTME. Citation Format: Amelie Daugherty-Lopes, Eva Perez-Guijarro, Vishaka Gopalan, Jessica Rappaport, Quanyi Chen, April Huang, Khiem C. Lam, Sung Chin, Jessica Ebersole, Emily Wu, Gabriel Needle, Isabella Church, George Kyriakopoulos, Shaojun Xie, Yongmei Zaho, Charli Gruen, Antonella Sassano, Romina E Araya, Andres Thorkelsson, Cari Smith, Maxwell P. Lee, Sridhar Hannenhalli, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid, Shaojun Xie. Dissecting the brain metastatic microenvironment to uncover immune and molecular correlates of response to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A002.

Abstract A025: RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and malignant type of ovarian cancer with a high frequency of p53 mutation and hyperactivation of the AKT pathway. Even though roughly 85% of patients achieve remission after initial surgical debulking and chemotherapy, four out of five HGSOC patients will experience disease recurrence, resulting in a 5-year survival rate of 43%. We have identified and characterized two TAp63 Regulated Oncogenic Long non-coding RNAs (TROLL-2 and TROLL-3) whose transcription is induced by mutant p53 and high expression level correlate with a more aggressive ovarian cancer grade and phosphorylation of AKT. In-depth analysis demonstrated that TROLL-2 and TROLL-3 directly activate AKT in tumor cells, thus linking p53 alterations to AKT activity. The use of AKT inhibitors in the clinic is limited because of frequent adverse effects, likely due to the constitutive expression of AKT in all cells. TROLL-2 and TROLL-3 expression is limited to tumor cells and provides a more specific target for decreasing AKT activation, potentially minimizing off-target effects. We have optimized reduction of TROLL-2 and TROLL-3 expression using antisense oligonucleotides (ASOs) in an ex vivo perfusion platform for 3D culture of microtumors from primary patient derived tumors. Samples show maximum reduction of proliferation (EdU) and phosphorylation of AKT when TROLL-2 and TROLL-3 ASOs are used concurrently. We also performed phospho-proteomics and single cell RNAseq to compare individual ASO-mediated knockdown of each lncRNA to combined targeted ASO treatments. We have developed primary patient derived xenograft models using ovarian tumors orthotopically implanted into NSG mice generating a relevant preclinical model to determine the efficacy of ASO treatment. To reduce the rapid degradation and clearance of ASOs in circulation, ASOs were encapsulated into lipid nanoparticles and metal-organic frameworks (MOFs) and we are currently testing these in vivo. Since AKT activation is associated with increased staging and resistance to platinum and targeted therapies in ovarian cancer patients, our data provides preclinical rationale for the implementation of ASOs and the use of new delivery methods as a relevant translational therapy and novel method to exploit the specificity of TROLL-2 and TROLL-3 expression in HGSOC. Citation Format: Ashley Lui, Marco Napoli, Jaden R Baldwin, Christina L Carr, Angie Rivera, Duy Nguyen, W. Gregory Sawyer, Michael R Dunne, Erin George, Omar K Farha, Michelle Teplensky, Elsa R Flores. RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A025.

Lymph Node Inclusion in a Modified Osteomyocutaneous Allograft for Vascularized Composite Allotransplantation: Establishment and Feasibility Assessment in a Pig Model.

Representative translational animal models play a key role in vascularized composite allotransplantation (VCA) research. A composite porcine hindlimb flap, previously described, is a relevant preclinical model. However, its bulkiness and the absence of critical immunologic tissues make it less suitable for investigating the unique immunologic features of VCA. We aimed to further develop this model by reducing its bulkiness and by including donor-draining lymph nodes. We conducted an anatomic study by harvesting 11 porcine osteomyocutaneous flaps (4 conventional and 7 modified techniques), which were characterized by computed tomography. Furthermore, 8 allotransplantations were performed in Swiss landrace pigs. After the procedure, animals were assigned to a model development and control group (N = 4 per group). No immunosuppression was given, and animals were followed up until grade 3 rejection. With the modified technique, the flap weight was significantly reduced with a mean weight of 831 g, corresponding to 1.8% total body weight versus 1710 g in the conventional technique, representing 4.2% of total body weight (P < 0.0001). The muscle/bone ratio was reduced from 8.24 (conventional) to 2.92 (modified), (P = 0.03). Histologically, graft-draining lymph nodes showed typical changes related to rejection and no signs of ischemia after in vivo transplantation. By modifying the surgical technique, the bulkiness of the flap was markedly reduced, without impairing its vascularization and reliably including vascularized graft-draining lymph nodes. Our modified VCA model in the pig presents distinct advantages for surgery as well as immunologic analysis, warranting a large-scale use for experimental reconstructive transplantation studies.

Robotic manipulation of cardiomyocytes to identify gap junction modifiers for arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a leading cause of sudden cardiac death among young adults. Aberrant gap junction remodeling has been linked to disease-causative mutations in plakophilin-2 (PKP2). Although gap junctions are a key therapeutic target, measurement of gap junction function in preclinical disease models is technically challenging. To quantify gap junction function with high precision and high consistency, we developed a robotic cell manipulation system with visual feedback from digital holographic microscopy for three-dimensional and label-free imaging of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The robotic system can accurately determine the dynamic height changes in the cells' contraction and resting phases, microinject drug-treated healthy and diseased iPSC-CMs in their resting phase with constant injection depth across all cells, and deposit a membrane-impermeable dye that solely diffuses between cells through gap junctions for measuring the gap junction diffusion function. The robotic system was applied toward a targeted drug screen to identify gap junction modulators and potential therapeutics for ACM. Five compounds were found to dose-dependently enhance gap junction permeability in cardiomyocytes with PKP2 knockdown. In addition, PCO 400 (pinacidil) reduced beating irregularity in a mouse model of ACM expressing mutant PKP2 (R735X). These results highlight the utility of the robotic cell manipulation system to efficiently assess gap junction function in a relevant preclinical disease model, thus providing a technique to advance drug discovery for ACM and other gap junction-mediated diseases.

Synthesizing regulatory guidance for demonstrating preclinical efficacy and translating promising cell therapies to early phase clinical trials: a scoping review

BackgroundRegulatory applications for cell therapy face more objections compared to conventional small molecule or biological drugs, leading to delays in market approval and clinical adoption. Increased regulatory objections frequently relate to issues regarding preclinical evidence, such as experimental design of animal studies, selection of animal models, endpoints, and determination of mechanism of action. Synthesis and clarification of the preclinical evidence necessary to demonstrate treatment efficacy and advance into early-phase clinical trials is needed to help researchers avoid regulatory objections.MethodsWe conducted a scoping review in which we searched repositories of the International Council for Harmonisation and all national member organizations (N = 38) for documents related to preclinical studies of cell therapies. Active guidance documents related to cell therapy were included, with no restrictions based on the year or language of publication. Data extraction was conducted in duplicate with conflicts resolved through consensus discussion.ResultsFrom 1215 identified documents, a total of 182 were included and analyzed, with 71% originating from ten major regulatory agencies. The most prevalent preclinical item addressed was the mechanism of action (n = 161, 88% of documents), underscoring its importance in bridging preclinical findings to clinical application. Most documents (n = 140, 77%) emphasized the importance of using clinically relevant preclinical models, though specific recommendations on models of disease were less common (n = 81, 45%). Selection of clinically relevant intervention parameters (n = 136, 75%) and outcome measures (n = 121, 66%) were also frequently recommended, but selection of relevant comparator groups appeared less frequently (n = 35, 19%). Furthermore, robust study design elements such as randomization and blinding were less frequently recommended, appearing in 31% of documents (n = 57). Comparison with clinical trial guidance revealed a significant gap in the rigor of study design recommendations for preclinical research.ConclusionsRegulatory guidance for preclinical efficacy studies often recommends a strong emphasis on the clinical relevance of animal models, intervention parameters, outcomes, and mechanism of action. Incorporating these recommendations into early preclinical studies should improve the acceptability of preclinical evidence for approval by the relevant national regulators and can be used as a guide to ensure that all evidence that regulators say they expect is efficiently assembled into new clinical trial applications.

Sensory-Motor Neuropathy in Mfn2 T105M Knock-in Mice and Its Reversal by a Novel Piperine-Derived Mitofusin Activator.

Mitochondrial dysfunction is a hallmark of many genetic neurodegenerative diseases, but therapeutic options to reverse mitochondrial dysfunction are limited. While recent studies support the possibility of improving mitochondrial fusion/fission dynamics and motility to correct mitochondrial dysfunction and resulting neurodegeneration in Charcot-Marie-Tooth disease (CMT) and other neuropathies, the clinical utility of reported compounds and relevance of preclinical models are uncertain. Here, we describe motor and sensory neuron dysfunction characteristic of clinical CMT type 2 A in a CRISPR/Casp-engineered Mfn2 Thr105Met (T105M) mutant knock-in mouse. We further demonstrate that daily oral treatment with a novel mitofusin activator derived from the natural product piperine can reverse these neurologic phenotypes. Piperine derivative 8015 promoted mitochondrial fusion and motility in Mfn2-deficient cells in a mitofusin-dependent manner and reversed mitochondrial dysfunction in cultured fibroblasts and reprogrammed motor neurons from a human CMT2A patient carrying the MFN2 T105M mutation. Like previous mitofusin activators, 8015 exhibited stereospecific functionality, but the more active stereoisomer, 8015-P2, is unique in that it has subnanomolar potency and undergoes entero-hepatic recirculation which extends its in vivo half-life. Daily administration of 8015-P2 to Mfn2 T105M knock-in mice for 6 weeks normalized neuromuscular and sensory dysfunction and corrected histological/ultrastructural neurodegeneration and neurogenic myoatrophy. These studies describe a more clinically relevant mouse model of CMT2A and an improved mitofusin activator derived from piperine. We posit that 8015-P2 and other piperine derivatives may benefit CMT2A or other neurodegenerative conditions wherein mitochondrial dysdynamism plays a contributory role. SIGNIFICANCE STATEMENT: Mitochondrial dysfunction is widespread and broadly contributory in neurodegeneration, but difficult to target therapeutically. Here, we describe 8015-P2, a new small molecule mitofusin activator with ∼10-fold greater potency and improved in vivo pharmacokinetics versus comparators, and demonstrate its rapid reversal of sensory and motor neuron dysfunction in an Mfn2 T105M knock-in mouse model of Charcot-Marie-Tooth disease type 2 A. These findings further support the therapeutic approach of targeting mitochondrial dysdynamism in neurodegeneration.

IMMUNE AND MOLECULAR CORRELATES OF RESPONSE TO IMMUNOTHERAPY REVEALED BY BRAIN-METASTATIC MELANOMA MODELS.

Despite the promising results of immune checkpoint blockade (ICB) therapy, outcomes for patients with brain metastasis (BrM) remain poor. Identifying resistance mechanisms has been hindered by limited access to patient samples and relevant preclinical models. Here, we developed two mouse melanoma BrM models that recapitulate the disparate responses to ICB seen in patients. We demonstrate that these models capture the cellular and molecular complexity of human disease and reveal key factors shaping the tumor microenvironment and influencing ICB response. BR1-responsive tumor cells express inflammatory programs that polarize microglia into reactive states, eliciting robust T cell recruitment. In contrast, BR3-resistant melanoma cells are enriched in neurological programs and exploit tolerance mechanisms to maintain microglia homeostasis and limit T cell infiltration. In humans, BR1 and BR3 expression signatures correlate positively or negatively with T cell infiltration and BrM patient outcomes, respectively. Our study provides clinically relevant models and uncovers mechanistic insights into BrM ICB responses, offering potential biomarkers and therapeutic targets to improve therapy efficacy.

Pre-operative environmental enrichment does not yield a prophylactic effect against traumatic brain injury-induced neurobehavioral deficits

The robustness of environmental enrichment (EE) in ameliorating neurobehavioral and cognitive deficits after experimental traumatic brain injury (TBI) is unequivocal. What is equivocal is whether EE can function as a prophylactic to afford resiliency and neuroprotection against TBI. We hypothesized that pre-operative EE would yield a protective effect against TBI-induced motor, cognitive, and coping deficits, and that further improvements would be conferred when EE is provided before and after TBI. To test the hypotheses, adult male rats received either 4 weeks of EE or standard (STD) housing prior to undergoing a controlled cortical impact of moderate severity (2.8 mm deformation at 4 m/s) or sham injury while under anesthesia. After injury, the rats were randomly assigned to post-operative EE or STD housing. Motor ability, spatial learning, and memory retention were assessed by beam-walk and water maze tests, respectively. Active and passive behavioral coping strategies were evaluated with the shock probe defensive burying (SPDB) test. c-Fos and cortical lesion volume were also quantified. The post-TBI enrichment groups (EE + TBI + EE and STD + TBI + EE) did not differ (p > 0.05) and performed better than the post-TBI STD-housed groups (EE + TBI + STD and STD + TBI + STD) on motor and cognition (p < 0.05). The post-TBI STD groups did not differ, regardless of whether in EE or STD living conditions before injury (p > 0.05). Moreover, both post-TBI enrichment groups performed better in the SPDB test relative to the STD + TBI + STD group (p < 0.05). c-Fos + cells were upregulated in the ipsilateral CA1 in both pre-injury EE groups relative to the pre-injury STD groups (p < 0.05). No statistical differences were observed in cortical lesion volume among the groups. Overall, these data do not support the hypothesis as no neuroprotective effect was observed with 4 weeks of pre-operative EE and no additional benefit was achieved in the TBI group receiving both pre-and-post EE relative to the TBI group receiving only post-EE. However, the data do reinforce the consistency of post-TBI EE in producing robust neurobehavioral benefits, which further supports this paradigm as a relevant preclinical model of neurorehabilitation.

Inflammation in Dry Eye Disease-Pathogenesis, Preclinical Animal Models, and Treatments.

Dry eye disease (DED) is a rapidly growing ocular surface disease with a significant socioeconomic impact that affects the patients' visual function and, thus, their quality of life. It is distinguished by a loss of tear film homeostasis, leading to tear film instability, hyperosmolarity, ocular surface inflammation, and neurosensory abnormalities, with all of these playing etiological roles in the propagation of the vicious DED circle. While current treatments primarily focus on reducing tear film instability and hyperosmolarity, increasingly more attention is being placed on tackling the underlying inflammation that propagates and potentiates these factors. As such, preclinical models are crucial to further elucidate the DED pathophysiology and develop novel therapeutic strategies. This review outlines the role of inflammation in DED, highlighting related signs and diagnostic tools before focusing on relevant preclinical animal models and potential therapeutic strategies to tackle DED-associated inflammation.

Syngeneic mouse model of YES-driven metastatic and proliferative hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a disease of high unmet medical need that has become a global health problem. The development of targeted therapies for HCC has been hindered by the incomplete understanding of HCC pathogenesis and the limited number of relevant preclinical animal models. We recently unveiled a previously uncharacterized YES kinase (encoded by YES1)-dependent oncogenic signaling pathway in HCC. To model this subset of HCC, we established a series of syngeneic cell lines from liver tumors of transgenic mice expressing activated human YES. The resulting cell lines (referred to as HepYF) were enriched for expression of stem cell and progenitor markers, proliferated rapidly, and were characterized by high SRC family kinase (SFK) activity and activated mitogenic signaling pathways. Transcriptomic analysis indicated that HepYF cells are representative of the most aggressive proliferation class G3 subgroup of HCC. HepYF cells formed rapidly growing metastatic tumors upon orthotopic implantation into syngeneic hosts. Treatment with sorafenib or the SFK inhibitor dasatinib markedly inhibited the growth of HepYF tumors. The new HepYF HCC cell lines provide relevant preclinical models to study the pathogenesis of HCC and test novel small-molecule inhibitor and immunotherapy approaches.

Leveraging interindividual variability in threat conditioning of inbred mice to model trait anxiety.

Trait anxiety is a major risk factor for stress-induced and anxiety disorders in humans. However, animal models accounting for the interindividual variability in stress vulnerability are largely lacking. Moreover, the pervasive bias of using mostly male animals in preclinical studies poorly reflects the increased prevalence of psychiatric disorders in women. Using the threat imminence continuum theory, we designed and validated an auditory aversive conditioning-based pipeline in both female and male mice. We operationalised trait anxiety by harnessing the naturally occurring variability of defensive freezing responses combined with a model-based clustering strategy. While sustained freezing during prolonged retrieval sessions was identified as an anxiety-endophenotype biomarker in both sexes, females were consistently associated with an increased freezing response. RNA-sequencing of CeA, BLA, ACC, and BNST revealed massive differences in phasic and sustained responders' transcriptomes, correlating with transcriptomic signatures of psychiatric disorders, particularly post-traumatic stress disorder (PTSD). Moreover, we detected significant alterations in the excitation/inhibition balance of principal neurons in the lateral amygdala. These findings provide compelling evidence that trait anxiety in inbred mice can be leveraged to develop translationally relevant preclinical models to investigate mechanisms of stress susceptibility in a sex-specific manner.

Understanding and Modeling the Pathophysiology of Hydrocephalus: In Search of Better Treatment Options

Hydrocephalus is caused by an overproduction of cerebrospinal fluid (CSF), an obstruction of fluid movement, or improper reabsorption. CSF accumulation in the brain’s ventricles causes ventriculomegaly, increased intracranial pressure, inflammation, and neural cell injury. Hydrocephalus can arise from brain trauma, hemorrhage, infection, tumors, or genetic mutations. Currently, there is no cure for hydrocephalus. Treatments like shunting and endoscopic third ventriculostomies are used, but, unfortunately, these therapeutic approaches require brain surgery and have high failure rates. The choroid plexus epithelium (CPe) is thought to be the major producer of CSF in the brain. It is a polarized epithelium that regulates ion and water movement from a fenestrated capillary exudate to the ventricles. Despite decades of research, control of electrolyte movement in the CPe is still not fully understood. This review discusses important transporters on the CPe, how some of these are regulated, and which of them could be potential targets for hydrocephalus treatment. To advance the development of hydrocephalus treatments, physiologically relevant preclinical models are crucial. This review covers some of the current animal and cell culture methods used to study hydrocephalus and highlights the need to develop standardized preclinical models that are used by multiple investigators in order to replicate critical findings and resolve controversies regarding potential drug targets.

Abstract LB364: Generation and Application of a Robust Humanized Mouse Tumor Model System for Evaluation of NK-Targeting Therapeutics

Abstract With the new waves of anti-tumor immunotherapy development, novel immunotherapies based on NK cells have emerged and advanced rapidly. Considering the inherent mechanisms of NK cells, various strategies are employed in tumor immunotherapy to maximize the efficacy of NK cells, including the following: 1) immune checkpoint inhibitors to relieve NK cell inhibition signals; 2) activation of NK cell activity; 3) targeted antibody therapy for tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC), and 4) cell therapy, such as CAR-NK. Although numerous NK-targeting immune therapeutics or various NK-modulating modalities have been developed or advanced toward clinical trials, there is a lack of clinically relevant preclinical animal models that can be widely accepted for the evaluation of NK-modulating agents, particularly for models that involve functional human NK cells in the test system. This study generated a humanized mouse tumor model system which involves specific reconstitution of functional and durable human NK cells in a murine system, providing a clinically relevant tumor microenvironment. We first engineered a stable monoclonal feeder cell line in vitro to expand NK cells isolated from human peripheral blood mononuclear cells (hPBMCs) using magnetic bead separation. Using this approach, the human primary NK cells were robustly and consistently amplified (up to hundreds of fold) while maintaining their purity and functionality. When compared with primary NK cells using in vitro immune assays including ADCC and FACs, the expanded NK cells showed comparable results as the parental cells. In vivo experimental results indicated that a durable and functional NK reconstitution can be established in tumor-bearing severely immune deficient mice expressing hIL-15. Furthermore, this NK-humanized tumor model system was proven to respond to standard of care (SOC) NK-targeting drugs. In summary, this novel NK humanized in vivo model may represent an effective preclinical tool for assessing NK-modulating drugs, with the hope of facilitating the development process of novel immune therapeutics employing both adaptive and innate immunity. Citation Format: Feng Lu, Weiqun Ding, Guangliang Yang, Yaoqing Jia, Zhaoxiang Wang, Xusheng Yuan, Ming Yin, Zhijian Yang, WenQing Yang. Generation and Application of a Robust Humanized Mouse Tumor Model System for Evaluation of NK-Targeting Therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB364.

Mitophagy modulation for the treatment of cardiovascular diseases.

Defects of mitophagy, the selective form of autophagy for mitochondria, are commonly observed in several cardiovascular diseases and represent the main cause of mitochondrial dysfunction. For this reason, mitophagy has emerged as a novel and potential therapeutic target. In this review, we discuss current evidence about the biological significance of mitophagy in relevant preclinical models of cardiac and vascular diseases, such as heart failure, ischemia/reperfusion injury, metabolic cardiomyopathy and atherosclerosis. Multiple studies have shown that cardiac and vascular mitophagy is an adaptive mechanism in response to stress, contributing to cardiovascular homeostasis. Mitophagy defects lead to cell death, ultimately impairing cardiac and vascular function, whereas restoration of mitophagy by specific compounds delays disease progression. Despite previous efforts, the molecular mechanisms underlying mitophagy activation in response to stress are not fully characterized. A comprehensive understanding of different forms of mitophagy active in the cardiovascular system is extremely important for the development of new drugs targeting this process. Human studies evaluating mitophagy abnormalities in patients at high cardiovascular risk also represent a future challenge.

Abstract 2841: Development and validation of a systemic human multiple myeloma model utilizing luciferase expressing MM1.S tumor cells

Abstract Multiple Myeloma (MM) is a complex hematologic malignancy characterized by clonal proliferation of transformed plasma cells leading to overproduction of monoclonal immunoglobulins and subsequently end-stage organ damage. Despite therapeutic advances, MM is an incurable disease with poor prognosis in high-risk patients, and thus relevant preclinical models are necessary to develop novel treatment strategies. We developed a Multiple Myeloma in vivo model by systemic implantation of the luciferase-expressing glucocorticoid sensitive tumor cell line MM1.S (MM1.S-Luc) into immunodeficient NCG mice. First, we generated a MM1.S-Luc monoclonal cell line from a polyclonal pool of stable luciferase-expressing cells by limiting dilution. Single cells from the stable pool were expanded under selective pressure and four MM1.S-Luc monoclonal lines were selected based on in vitro growth properties and bioluminescence intensity. Next, we determined the in vivo tumor growth profile by testing the monoclonal lines at two different cell inoculums, and disease progression was monitored by in vivo serial bioimaging. MM1.S-Luc tumor growth kinetics revealed a long latency and based on our in vivo imaging results, the tumor preferentially localized to the long bones, lungs, and mandible. For validation of the MM1.S-Luc in vivo model, we determined the efficacy and survival benefit in response to various clinically relevant standard of care agents including Dexamethasone, Daratumumab, Cyclophosphamide, Vincristine, Panobinostat, and Bortezomib. The quantification of tumor burden by bioluminescent imaging showed reduced tumor burden and prolonged survival with Panobinostat and Cyclophosphamide. In summary, we have developed a systemic MM1.S-Luc model that recapitulates the disease dissemination and invasiveness of multiple myeloma for evaluating novel therapeutic approaches to treatment. Citation Format: Bincy John, Christopher Currie, Nicole Westrick, Tyler Rowe, Andrew Wong, Elizabeth Rainbolt, Zachary Ward, Chassidy Hall, David Harris. Development and validation of a systemic human multiple myeloma model utilizing luciferase expressing MM1.S tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2841.

Abstract 6902: Development of a stable luciferase expressing PDX-derived cell line for translational in vitro and in vivo testing

Abstract Despite much effort invested in developing novel cancer therapies, more than 90% of clinical studies fail due to toxicity or lack of efficacy in patients. This can mainly be ascribed to lack of relevant and translational preclinical models. A common model used in preclinical studies is the xenograft model using immortalized cancer cells. Cell line-based models are cost efficient and can be genetically manipulated but are often criticized for lacking translational value. A murine model with higher clinical relevance is the patient derived xenografts (PDX) model established by implanting patients’ tumor tissue onto immunodeficient mice. Knowledge of patient therapy response, mutational status and tumor heterogeneity makes it a relevant model. However, PDX models are costly, not applicable for in vitro assays or subjective to genetic manipulation. To more closely mimic in vivo tumors we have established a PDX-derived cell line (PDXc) and characterized it extensively in a head-to-head comparison to the parental PDX model. Cells were isolated from a subcutaneous (SC) glioblastoma (GBM) PDX tumor (ST146), processed into single cells and cultivated in vitro. Once the PDXc were stable over several passages the cells were re-implanted into mice. Tumor take and growth rate were evaluated against the parental PDX model and common characteristics of the model such as EFGR and Vimentin expression were determined by IHC. We furthermore generated a stable Luciferase expressing PDXc using 3rd generation lentiviral particles and implanted the cells in an intracranial (IC) model of GBM. Tumor growth was evaluated using bioluminescence imaging (BLI) and MRI. Upon multiple passages and freeze cycles the cell line (ST146c) exhibited a stable in vitro doubling time (2.6 ± 0.2 days) with a morphology like other GBM cell lines with formation of neurospheres. STR analysis confirmed a genetic identical profile to ST146. ST146c implanted into mice had similar tumor take and growth rates to the parental PDX when injected IC (&amp;gt;90%, Td=3 d) and SC (&amp;gt;75%, Td=6 d). We also showed that ST146c had more homogeneous tumor growth. Ex vivo analysis showed similar expressions of EGFR (-), Vimentin (+++) and Ki67 (++) between the models. The BLI signal obtained from luciferase expressing ST146c implanted orthotopically was correlated to tumor volume obtained by MRI. We developed a PDX cell line that preserved critical in vitro and in vivo characteristics of the PDX model such as doubling time, tumor take rate and marker expression making it feasible to use the PDXc model in future preclinical studies. This will allow in vitro screens with patient derived tumor cells. Furthermore, the expression of luciferase in the PDXc enables intracranial and metastatic implantation with tumor monitoring using BLI. This platform consisting of in vitro and in vivo PDX models could be used as a predictive translational platform for testing new cancer therapies. Citation Format: Sigrid Cold, Rikke N. Bøge, Emma Papin, Ida T. Michler, Lotte K. Kristensen, Trine B. Engel, Andreas Kjaer, Carsten H. Nielsen, Sebastian Gnosa. Development of a stable luciferase expressing PDX-derived cell line for translational in vitro and in vivo testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6902.

Abstract 3392: Generation and characterization of transgenic oncopig model for lung cancer

Abstract Lung cancer (LC) is the leading cause of cancer-associated death in the United States. Preclinical findings in small animal models do not translate into human clinics. There is an urgent need to develop relevant preclinical large animal models for LC. We describe a novel method to induce LC in transgenic pigs carrying Cre-inducible KrasG12D and TrP53R167H mutations (Oncopigs). The Oncopigs (n=12; 9 females, and 3 males) at 9 weeks of age were anesthetized and adenovirus (1 × 1011 PFU) carrying Cre recombinase gene (Ad-Cre) ± polybrene (1:100) ± IL-8 (5 ng/ml) was injected via flexible bronchoscopy with two techniques: (1) Bronchial lavage (n=6) and (2) Transbronchial needle injection (n=6) into different lung lobes. As controls, a combination of polybrene ± IL-8 was injected without AdCre into the contralateral lobes in the same pigs (n=8). Oncopigs were monitored via clinical monitoring, blood counts, basic metabolic panel, and contrast-enhanced computed tomography (CT) imaging for LC progression and metastasis at 3-, 4-, 7-, 10-, 16-, and 26 weeks post-intervention. All animals tolerated the injections with expected weight gains. Blood counts and metabolic panels remained normal during LC progression. At the site of injection/lavage, lung masses were detected on CT imaging at 3 weeks post-injections. However, a decrease in masses was observed on CT at 7 weeks post-intervention. The decrease in size of the lung masses coincided with increasing attenuation. No distant metastases were observed on CT imaging. Lavage-based lesions appeared to be more heterogeneous with surrounding inflammatory changes. Transbronchial-injection-based lesions appeared more consolidated and coin-shaped without surrounding inflammatory changes. Control injection sites did not show any mass growth on CT imaging. Immunohistochemistry analysis on lung tumors revealed increased pancytokeratin, trichrome, E-cad, Ki-67, IBA, and CD3 expression. Further, immunofluorescence revealed decreased E-Cad, CK-19, and Zo-1 expression, whereas increased expression of N-Cad, FN1, snail, CD44, α-SMA, and γ-catenin, was observed in the LC and metastatic tissue compared to normal. We successfully generated and characterized a novel transgenic Oncopig LC model. There is robust tumor production status post AdCre delivery. One of four Oncopigs had carcinomatous metastatic skin growth. The remaining 7 Oncopigs are monitored longitudinally for tumor growth and development of metastasis. Citation Format: Kirtan Joshi, Nagabhishek Sirpu, Amanda Schmelzle, Ravikanth Poonooru, Benjamin Nelson, Pradeep Subramanyam, Colleen Garrett, Mariana Sponchiado, Sarah Schlink, Samantha Gerb, Jesse Porter, Dae Kim, Jeffrey Kunin, Jeffrey Bryan, Timothy Hoffman, Bhanu Telugu, Jussuf Kaifi, Satyanarayana Rachagani. Generation and characterization of transgenic oncopig model for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3392.

Abstract 2360: ADV101, a first-in-class IL-1RAP ADC demonstrates significant anti-tumor activity in solid tumor models

Abstract Chronic inflammation is intricately linked to cancer development, and the IL-1 cytokine family stands out as pivotal regulators of inflammation. All the pro-inflammatory cytokines within the IL-1 family rely on a single co-receptor called IL-1RAP which therefore emerges as a crucial and central node for modulating these pathways. While healthy tissues, except for the placenta, showed minimal plasma membrane IL-1RAP expression, tumor tissues exhibited high IL-1RAP expression, particularly in Acute myeloid leukaemia, Ewing sarcoma, ovarian cancer, head and neck, and squamous gastric and oesophageal cancers. In addition, elevated IL-1RAP expression in haematological and solid malignancies has also been associated with poor overall survival, making it an attractive target for therapeutic intervention. In this study, we present the development and characterization of an antibody-drug-conjugate (ADC) targeting IL-1RAP for potential anti-tumor applications. Following on from this, an anti-IL-1RAP antibody, ADV58, was generated and confirmed to have the anticipated biophysical, specificity, and functional properties, including low nanomolar affinity (SPR and ELISA), selectivity/specificity to human IL-1RAP, and internalization potential into IL-1RAP-positive tumor cells. In addition, ADV58 bind similarly well to human, cynomolgus and rhesus monkey IL-1RAP antigens but not to rat and mouse. Altogether these properties made ADV58 an ideal candidate for an ADC program. Concurrently, solid tumor cell lines from indications of interest with elevated IL-1RAP expression were identified, laying the groundwork for disease relevant pre-clinical tumor models. Using these models, the anti-tumor potential of ADV101 (ADV58-derived ADC) was assessed in vitro and in vivo in successful proof-of-concept studies by conjugating ADV58 with monomethyl auristatin E (MMAE, DAR of 4) or Deruxtecan (DXd, DAR of 8). Humanisation and conjugation did not compromise binding of the naked antibody, the ADV58-MMAE or the ADV58-DXd ADCs to human and non-human primate IL-1RAP. In vitro, the ADCs selectively killed IL-1RAP-positive tumor cells. In vivo efficacy studies in IL-1RAP-positive subcutaneous xenograft tumor models also demonstrated a significant dose-dependent and IL-1RAP antigen-dependent anti-tumor response for both anti-IL-1RAP ADCs. Altogether the robust preclinical data package supports the continued development of ADV101 as a novel targeted ADC therapeutic strategy for a broad spectrum of cancers. Citation Format: Richard C. A. Sainson, Laura Demolis, Edouard De Dreuzy, Olivier Favre-Bulle, Alexis Collette, Lucas Bourhis, Soha Reda El Sayed, Delphine Genin, Clementine Primus, Lucie Bouquet, Ksenija Pavletic, Christophe Ferrand, Marina Deschamps, Stephane Lameynardie, Erik Rutjens, Jens Hasskarl. ADV101, a first-in-class IL-1RAP ADC demonstrates significant anti-tumor activity in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2360.