Preclinical Mouse Model Research Articles

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.

EXPRESS: 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 (HDACi) mocetinostat and the antimetabolite drug capecitabine in triple-negative mammary neoplasms in a preclinical mouse model. 35 female mice were grouped into the control group, capecitabine group, mocetinostat group, and combine drugs group. At the end of experimental period, body weight, 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 mosetinostat 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.

Identifying Plasmodium P36 and P52 antigens for coadministration with circumsporozoite protein to enhance vaccine efficacy

Vaccines targeting the complex pre-erythrocytic stage of Plasmodium parasites may benefit from the inclusion of multiple antigens. However, discerning protective effects can be difficult because newer candidates may not be as protective as leading antigens like the circumsporozoite protein (CSP) in the conventional pre-clinical mouse model. We developed a modified mouse model challenge strategy that maximizes the contribution of T cells induced by novel candidate antigens at the sporozoite challenge time point and used this approach to test Plasmodium P36 and P52 vaccine candidates alone and in concert with non-protective doses of CSP. Co-administration of P36 and/or P52 with CSP achieved 80-100% sterile protection in mice, compared to only 7-30% protection for each individual antigen. P36 and P52 vaccination induced murine CD4+ and CD8+ T cell responses, but not antibody responses. This study adds P36 and P52 as promising vaccine antigens that may enhance the protection achieved by CSP vaccination.

Timing of standard chow exposure determines the variability of mouse phenotypic outcomes and gut microbiota profile.

Standard chow diets influence reproducibility in animal model experiments because chows have different nutrient compositions, which can independently influence phenotypes. However, there is little evidence of the role of timing in the extent of variability caused by chow exposure. Here we measured the impact of different diets (5V5M, 5V0G, 2920X and 5058) and timing of exposure (adult exposure (AE), lifetime exposure (LE) and developmental exposure (DE)) on growth and development, metabolic health indicators and gut bacterial microbiota profiles across genetically identical C57BL/6J mice. Diet drove differences in macro- and micronutrient intake for all exposure models. AE had no effect on phenotypic outcomes. However, LE mice exhibited significant sex-dependent diet effects on growth, body weight and body composition. LE effects were mostly absent in the DE model, where mice were exposed to chow differences only from conception to weaning. Both AE and LE models exhibited similar diet-driven beta diversity profiles for the gut bacterial microbiota, with 5058 diet driving the most distinct profile. However, compared with AE, LE effects on beta diversity were sex dependent, and LE mice exhibited nine times more differentially abundant bacterial genera, the majority of which were inversely affected by 2920X and 5058 diets. Our findings demonstrate that LE to different chow diets has the greatest impact on the reproducibility of several experimental measures commonly used in preclinical mouse model studies. Importantly, weaning mice from different diets onto the same diet for maturation may be an effective way to reduce unwanted phenotypic variability among experimental models.

How exercise shapes the anti-inflammatory environment in multiple sclerosis – a conceptual framework focusing on tryptophan-derived molecules in T cell differentiation

Abstract Multiple Sclerosis (MS) is a chronic neuroinflammatory autoimmune characterized by inflammation-induced lesion formation after immune cell infiltration into the central nervous system. T cells play an intriguing role in MS immunopathology and research over the past decade has shown that tryptophan (TRP)-derived metabolites are crucial molecules affecting T cell differentiation, also in MS, and are modulated by exercise. The aryl hydrocarbon receptor (AHR), for which TRP metabolites are well-known ligands, has been elucidated as main driver of T cell differentiation and an enhanced anti-inflammatory cellular milieu in human MS and preclinical mouse models. By integrating evidence from different research fields, the aim of this article is to summarize and critically discuss the potential of exercise to activate the AHR in T cells by modulating circulating TRP-derived metabolites and to provide a conceptual framework on potential benefits in MS immunopathology.

A generative artificial intelligence approach for antibiotic optimization.

Antimicrobial resistance (AMR) poses a critical global health threat, underscoring the urgent need for innovative antibiotic discovery strategies. While recent advances in peptide design have yielded numerous antimicrobial agents, optimizing these molecules experimentally remains challenging due to unpredictable and resource-intensive trial-and-error approaches. Here, we introduce APEX Generative Optimization (APEX GO ), a generative artificial intelligence (AI) framework that integrates a transformer-based variational autoencoder with Bayesian optimization to design and optimize antimicrobial peptides. Unlike traditional supervised learning approaches that screen fixed databases of existing molecules, APEX GO generates entirely novel peptide sequences through arbitrary modifications of template peptides, representing a paradigm shift in peptide design and antibiotic discovery. Our framework introduces a new peptide variational autoencoder with design and diversity constraints to maintain similarity to specific templates while enabling sequence innovation. This work represents the first in vitro and in vivo experimental validation of generative Bayesian optimization in any setting. Using ten de-extinct peptides as templates, APEX GO generated optimized derivatives with enhanced antimicrobial properties. We synthesized 100 of these optimized peptides and conducted comprehensive in vitro characterizations, including assessments of antimicrobial activity, mechanism of action, secondary structure, and cytotoxicity. Notably, APEX GO achieved an outstanding 85% ground-truth experimental hit rate and a 72% success rate in enhancing antimicrobial activity against clinically relevant Gram-negative pathogens, outperforming previously reported methods for antibiotic discovery and optimization. In preclinical mouse models of Acinetobacter baumannii infection, several AI-optimized molecules-most notably derivatives of mammuthusin-3 and mylodonin-2-exhibited potent anti-infective activity comparable to or exceeding that of polymyxin B, a widely used last-resort antibiotic. These findings highlight the potential of APEX GO as a novel generative AI approach for peptide design and antibiotic optimization, offering a powerful tool to accelerate antibiotic discovery and address the escalating challenge of AMR.

Impaired spatial coding and neuronal hyperactivity in the medial entorhinal cortex of aged App NL-G-F mice.

The progressive accumulation of amyloid beta (Aβ) pathology in the brain has been associated with aberrant neuronal network activity and poor cognitive performance in preclinical mouse models of Alzheimer's disease (AD). Presently, our understanding of the mechanisms driving pathology-associated neuronal dysfunction and impaired information processing in the brain remains incomplete. Here, we assessed the impact of advanced Aβ pathology on spatial information processing in the medial entorhinal cortex (MEC) of 18-month App NL-G-F/NL- G-F knock-in (APP KI) mice as they explored contextually novel and familiar open field arenas in a two-day, four-session recording paradigm. We tracked single unit firing activity across all sessions and found that spatial information scores were decreased in MEC neurons from APP KI mice versus those in age-matched C57BL/6J controls. MEC single unit spatial representations were also impacted in APP KI mice. Border cell firing preferences were unstable across sessions and spatial periodicity in putative grid cells was disrupted. In contrast, MEC border cells and grid cells in Control mice were intact and stable across sessions. We then quantified the stability of MEC spatial maps across sessions by utilizing a metric based on the Earth Mover's Distance (EMD). We found evidence for increased instability in spatially-tuned APP KI MEC neurons versus Controls when mice were re-exposed to familiar environments and exposed to a novel environment. Additionally, spatial decoding analysis of MEC single units revealed deficits in position and speed coding in APP KI mice in all session comparisons. Finally, MEC single unit analysis revealed a mild hyperactive phenotype in APP KI mice that appeared to be driven by narrow-spiking units (putative interneurons). These findings tie Aβ-associated dysregulation in neuronal firing to disruptions in spatial information processing that may underlie certain cognitive deficits associated with AD.

Revisited guidelines for metabolic tolerance tests in mice.

Preclinical mouse models are extensively used in biomedical research to gain insight into disease mechanisms and to test new drug treatments. Glucose and insulin tolerance tests are simple experimental tests frequently used worldwide to assess glucose metabolism in mice. Various guidelines and methodological considerations have been published to help researchers standardize procedures and optimize research outcomes. Yet, there is still important experimental heterogeneity in the way these simple procedures are performed, with no real consensus on what the best practices are to achieve high-quality research and reproducible results. Here we critically examine several published guidelines and recent technical reports on how to perform these metabolic tests in laboratory mice and discuss the influence of various confounding factors on test results. We hope this work will help scientists establish more consensual guidelines for maximizing the relevance and clinical translation of studies using mouse models in metabolic research.

Reduced cross-protective potential of Omicron compared to ancestral SARS-CoV-2 spike vaccines against potentially zoonotic coronaviruses

The COVID-19 pandemic has emphasised the importance of vaccines and preparedness against viral threats crossing species barriers. In response, a worldwide vaccination campaign targeting SARS-CoV-2 was implemented, which provides some cross-protective immunological memory to other coronavirus species with zoonotic potential. Following a vaccination regimen against SARS-CoV-2 spike in a preclinical mouse model, we were able to demonstrate the induction of neutralizing antibodies towards multiple human ACE2 (hACE2)-binding Sarbecovirus spikes. Importantly, compared to vaccines based on the SARS-CoV-2 Reference strain, vaccines based on Omicron spike sequences induced drastically less broadly cross-protective neutralizing antibodies against other hACE2-binding sarbecoviruses. This observation remained true whether the vaccination regimens were based on protein subunit or mRNA / LNP vaccines. Overall, while it may be necessary to update vaccine antigens to combat the evolving SARS-CoV-2 virus for enhanced protection from COVID-19, Reference-based vaccines may be a more valuable tool to protect against novel coronavirus zoonoses.

Tumor cell-derived spermidine promotes a pro-tumorigenic immune microenvironment in glioblastoma via CD8+ T cell inhibition.

The glioblastoma (GBM) microenvironment is enriched in immunosuppressive factors that potently interfere with the function of cytotoxic T lymphocytes. Cancer cells can directly impact the immune system, but the mechanisms driving these interactions are not completely clear. Here we demonstrate that the polyamine metabolite spermidine (SPD) is elevated in the GBM tumor microenvironment. Exogenous administration of SPD drives tumor aggressiveness in an immune-dependent manner in pre-clinical mouse models via reduction of CD8+ T cell frequency and reduced cytotoxic function. Knockdown of ornithine decarboxylase, the rate-limiting enzyme in spermidine synthesis, did not impact cancer cell growth in vitro but did result in extended survival. Furthermore, glioblastoma patients with a more favorable outcome had a significant reduction in spermidine compared to patients with a poor prognosis. Our results demonstrate that spermidine functions as a cancer cell-derived metabolite that drives tumor progression by reducing CD8+ T cell number and function.

Integrating natural commensals and pathogens into preclinical mouse models.

Fundamental discoveries in many aspects of mammalian physiology have been made using laboratory mice as research models. These studies have been facilitated by the genetic tractability and inbreeding of such mice, the large set of immunological reagents that are available, and the establishment of environmentally controlled, high-throughput facilities. Such facilities typically include barriers to keep the mouse colonies free of pathogens and the frequent re-derivation of the mice severely limits their commensal flora. Because humans have co-evolved with microorganisms and are exposed to a variety of pathogens, a growing community of researchers posits that preclinical disease research can be improved by studying mice in the context of the microbiota and pathogens that they would encounter in the natural world. Here, we provide a perspective of how these different approaches can be combined and integrated to improve existing mouse models to enhance our understanding of disease mechanisms and develop new therapies for humans. We also propose that the term 'mice with natural microbiota' is more appropriate for describing these models than existing terms such as 'dirty mice'.

Abstract C041: MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer

Abstract Patients with castration-resistant prostate cancer (CRPC) are generally unresponsive to tumor targeted and immunotherapies. Whether genetic alterations acquired during the evolution of CRPC impact immune and immunotherapy responses is largely unknown. Using our innovative electroporation-based mouse models, we generated distinct genetic subtypes of CRPC found in patients and uncovered unique immune microenvironments. Specifically, mouse and human prostate tumors with MYC amplification and p53 disruption had weak cytotoxic lymphocyte infiltration and an overall dismal prognosis. MYC and p53 cooperated to induce tumor intrinsic secretion of VEGF, which by signaling through VEGFR2 expressed on CD8+ T cells, could directly inhibit T cell activity. Targeting VEGF-VEGFR2 signaling in vivo led to CD8+ T cell-mediated tumor and metastasis growth suppression and significantly increased overall survival in MYC and p53 altered CPRC. VEGFR2 blockade also led to induction of PD-L1, and in combination with PD-L1 immune checkpoint blockade produced anti-tumor efficacy in multiple preclinical CRPC mouse models. Thus, our results identify a genetic mechanism of immune suppression through VEGF signaling in prostate cancer that can be targeted to reactivate immune and immunotherapy responses in an aggressive subtype of CRPC. Citation Format: Katherine Murphy. MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer [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 C041.

A Pleiotropic Nanomedicine Mitigates Splenic Hyperplasia, Ineffective Erythropoiesis, G6PDH Anomaly through Redox Buffering in Preclinical Mice Model.

Here, we present a pleiotropic nanomedicine-a smart, functionalized redox buffering nanoparticle-that may be used to treat hematological diseases, associated splenic hyperplasia, and issues related to restricted erythropoiesis. With a diameter of 5-7 nm, the spherical nanomaterial is made of manganese oxide and citrate. Here, we have produced the novel nanomaterial and, using cutting-edge electron microscopic and spectroscopic techniques, extensively assessed its redox buffering potential in vitrowith its structural integrity. Using an appropriate animal model (phenyl hydrazine, PHz, intoxicated C57BL/6J mice), we assessed the therapeutic efficacy of the redox buffering nanomedicine in the treatment of anemia and related consequences. We have further investigated the intricate molecular mechanism of the nanomedicine and its therapeutic impact, which includes increased erythropoiesis and G6PDH production, decreased inflammatory responses, mitigation of splenic hyperplasia, and synergistic intracellular redox-buffering. To the best of our knowledge, our studies would find relevance in the innovative management of anemia, decreased erythropoiesis, and splenic hyperplasia.

Preclinical pharmacology characterization of HX009, a novel PD1 x CD47 Bi-specific antibody

Certain immune-checkpoint inhibitors have a narrow therapeutic window (TW) as cancer therapeutics, and engineered dual-/multi-targeting agents could potentially widen the TW to bring true clinical benefits. We report a new rationally-designed bispecific-antibody (BsAb), HX009, simultaneously targeting PD1 and CD47 to improve both the efficacy and safety over the respective single-targeting agents by grafting the extracellular domain of SIRPα onto the parental anti-PD1-monoclonal antibody, HX008. This resulted in an IgG4-based “2 × 2” symmetric structure but with an intentionally-reduced CD47-binding affinity, suggesting a novel candidate cancer immunotherapy. Specifically, HX009 has binding affinity constant of 8.951 × 10–9 M for human PD1 and 2.557 × 10–8 M for human CD47, respectively, where the CD47 binding is significantly weaker as compared to the binding affinity of HX008 to PD1 as well as the binding affinity of SIRPα-Fc to CD47, leading to little binding to RBCs and platelets and is contrasting to many CD47-agents in development. However, HX009 effectively and simultaneously binds to the PD1 and CD47 on PD1+CD47+ T-cells via cis-binding and elicits enhanced T cell activation compared to the parental HX008. HX009 caused little cytokine-release in human peripheral blood mononuclear cells. HX009 cross-species binds to cynomolgus monkey PD1/CD47 but not to rodents, making cynomolgus monkeys the choice of species to investigate the pharmacokinetics (PK) and toxicology of HX009. HX009’s anti-tumor activities were confirmed in several humanized preclinical mouse models by determining either its anti-PD1 (humanized hu-CD47-MC38 models) or anti-CD47 (HuT-102 lymphoma CDX and three PDX-AML models) functions, although limited available humanized models have hindered broadly demonstration of enhanced anti-tumor activities contributed from the dual targeting of the BsAb. The expanded DLBCL-PDX trial data suggested that both EBV-status and OX40 expression could potentially be two positive predictors for response to HX009. An intravenous (IV) infusion PK study in cynomolgus monkey revealed its largely vasculature distribution, terminal half-life (T1/2) of ~ 50 h, and dose-proportional exposure without accumulation. The anti-drug antibody (ADA) was observed in all monkeys as expected, affecting the PK parameters of repeated administration. The IV single-dose toxicology study with a 14-day observation revealed a maximum tolerated dose of 150 mg/kg, while the repeated-dose (once weekly for 4 weeks, 5 doses in total) study showed a highest non-severely toxic dose (HNSTD) of 15 mg/kg. The desired preclinical PK and safety profiles, along with its antitumor activity, support HX009’s candidacy for its clinical development.

Abstract B013: Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression

Abstract Breast cancer (BC) is the most diagnosed cancer and the second leading cancer-related cause of mortality among women in the US. Despite advances in treatment, BC remains a challenge due to its metastatic potential. The most aggressive BC subtype, triple negative (TN), often harbors mutations in the tumor suppressor gene, p53. One of the effects of mutant p53 involves the inhibition of the tumor and metastatic suppressive functions of TAp63, a p53 family member. Loss of TAp63 leads to the formation of metastatic mammary adenocarcinomas in mice, in part through the regulation of a group of nine long non-coding RNAs (lncRNAs), that we identified and named TAp63 regulated oncogenic lncRNAs (TROLLs). We previously characterized two TROLLs, TROLL-2 and TROLL-3, as markers of tumor progression in multiple cancer types via the activation of the AKT pathway. Currently we are investigating the role of another member, TROLL-9. To investigate the biological role of TROLL-9, we performed in vivo experiments using preclinical mouse models. Specifically, MCF-DCIS, MCF-CA1D, and MDA-MB-231 triple negative breast cancer (TNBC) cells constitutively expressing TROLL-9 were generated and utilized for two mouse models: orthotopic xenografts for primary tumor growth and spontaneous metastasis. The former was designed to identify the potential role of TROLL-9 in tumor formation, whereas the latter involved the resection of a primary tumor and monitoring for metastases via bioluminescence imaging. Notably, TROLL-9 overexpression led to increased formation of lung and livers metastases in all these models, suggesting a role for TROLL-9 in the metastatic cascade. In order to elucidate the mechanism of function of TROLL-9, two complementary approaches were used. Firstly, pulldown using MS2-TRAP followed by mass spectrometry (MS) was performed and led to the identification of putative protein interactors. In parallel, Laser Capture Microscopy (LCM) of the lung metastases followed by RNA-sequencing was used to assess for any gene expression changes due to TROLL-9 overexpression. Integration of the two datasets is currently being performed to unveil the network of genes and proteins perturbed by TROLL-9. Furthermore, to understand the relevance in BC patients, TROLL-9 expression levels were analyzed in the TCGA BC dataset. Interestingly, differences in TROLL-9 levels across BC subtypes were observed, thus supporting its putative role in TNBC. Overall, our data suggest a role for TROLL-9 in BC progression. Our findings provide evidence for further characterizing its mechanism of function as a potential therapeutic target for the treatment of invasive BC, particularly TNBC harboring TP53 mutations. Citation Format: Avani A Deshpande, Marco Napoli, John H Lockhart, Christina L Carr, Jaden R Baldwin, Elsa R Flores. Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression [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 B013.

Mechanisms driving epigenetic and transcriptional responses of microglia in a neurodegenerative lysosomal storage disorder model.

Lysosomal dysfunction is causally linked to neurodegeneration in many lysosomal storage disorders (LSDs) and is associated with various age-related neurodegenerative diseases 1,2 , but there is limited understanding of the mechanisms by which altered lysosomal function leads to changes in gene expression that drive pathogenic cellular phenotypes. To investigate this question, we performed systematic imaging, transcriptomic, and epigenetic studies of major brain cell types in Sgsh null (KO) mice, a preclinical mouse model for Sanfilippo syndrome (Mucopolysaccharidosis Type IIIA, MPS-IIIA) 3,4 . MPS-IIIA is a neurodegenerative LSD caused by homozygous loss-of-function (LoF) mutations in SGSH which results in severe early-onset developmental, behavioral, and neurocognitive impairment 5-15 . Electron microscopy, immunohistochemistry, and single-nucleus RNA-sequencing analysis revealed microglia as the cell type exhibiting the most dramatic phenotypic alterations in Sgsh KO mice. Further temporal analysis of microglia gene expression showed dysregulation of genes associated with lysosomal function and immune signaling pathways beginning early in the course of the disease. Sgsh deficiency similarly resulted in increases in open chromatin and histone acetylation at thousands of putative microglia-specific enhancers associated with upregulated genes but had much less impact on the epigenetic landscapes of neurons or oligodendrocytes. We provide evidence for dominant and context-dependent roles of members of the MITF/TFE family as major drivers of microglia-specific epigenetic and transcriptional changes resulting from lysosomal stress that are dependent on collaborative interactions with PU.1/ETS and C/EBP transcription factors. Lastly, we show that features of the transcriptomic and epigenetic alterations observed in murine Sgsh deficiency are also observed in microglia derived from mouse models of age-related neurodegeneration and in human Alzheimer's disease patients, revealing common and disease-specific transcriptional mechanisms associated with disease-associated microglia phenotypes.

DDEL-17. INTRA-TUMORAL CONVECTION ENHANCED DELIVERY OF DEXAMETHASONE REDUCES INFLAMMATORY SIGNATURES AND EXTENDS SURVIVAL IN GBM MODEL

Abstract Dexamethasone is the most widely utilized drug for glioblastoma (GBM). However, systemic delivery of dexamethasone in GBM patients is associated with significant side effects and increasing doses with worse survival. Convection-enhanced delivery (CED) can deliver high doses of immunotherapy directly into the tumor microenvironment (TME) while avoiding systemic toxicity. Here, we report that high doses of dexamethasone can be delivered locally by CED without side effects and increase survival in a GBM model along with reduced inflammatory myeloid signatures. We investigated CED of dexamethasone treatment on survival(n=40), adverse side effects, and the immunological TME through peripheral analyses, liquid chromatography–mass spectrometry, and histology in a preclinical syngeneic mouse model. Additionally, we assessed the transcriptional responses of human GBM slices and stem-cell-derived human microglia after steroid treatment through bulk and single-cell RNA sequencing. We found that 7-day treatment with CED of dexamethasone produced a significant survival advantage in glioma-bearing mice compared to non-treated control(p=0.03). Systemic dexamethasone achieved low levels of drug in the TME and caused dysregulated blood glucose, blood counts, and peripheral organ weights, while high CED doses avoided these side effects(p< 0.05 each). Steroid treatment of acute GBM slices and lipopolysaccharide-activated microglia in-vitro both reversed inflammatory myeloid signatures associated with poor survival and recurrence on RNA sequencing analyses. We demonstrate the preclinical efficacy of delivering high local doses of dexamethasone in a GBM model through CED and observed prolonged survival along with reduced adverse inflammatory myeloid signatures. No side effects were demonstrated after chronic CED treatment of dexamethasone while systemic delivery achieved poor tumor penetration and caused known hematologic and metabolic side effects supporting the potential to optimize the clinical use of this therapy. CED of dexamethasone provides us a new treatment paradigm to locally control inflammatory signatures in the glioma TME in a controlled fashion.

DDDR-04. PRECLINICAL INVESTIGATION OF A NOVEL BRAIN PENETRANT COMBINATION THERAPY TARGETING RAF AND MEK FOR MELANOMA BRAIN METASTASIS

Abstract INTRODUCTION Up to 75% of patients with advanced melanoma develop brain metastases. BRAF mutations are found in 50-55% of melanoma brain metastases. Current BRAF/MEK inhibitors, such as dabrafenib and vemurafenib, have shown responses on the order of 50-60% in patients with BRAF-mutant melanoma; however, responses are not durable. Better therapies are needed with improved penetration of the blood brain barrier. This study aims to evaluate the efficacy of two novel brain-penetrant inhibitors, KIN-7136 (MEK inhibitor) and KIN-8391 (RAF inhibitor), in preclinical mouse models of melanoma brain metastases driven by aberrant BRAF-MEK signaling. METHODS CellTiter-Glo® 2.0 Cell Viability Assay (Promega) was used for in vitro cell viability assays. Intracranial A375 melanoma models were generated in athymic nude mice to assess the pharmacodynamics, safety, and efficacy of combination therapy of KIN-7136 and KIN-8391. RESULTS In mice, the brain-to-plasma unbound partition coefficients (Kpuu) of KIN-7136 and KIN-8391 were 0.67 and 0.7 respectively, higher than the comparators binimetinib and belvarafenib, demonstrating improved brain exposure. The IC50s of KIN-7136/ KIN-8391 in melanoma cell lines were 27.0/36.5 nM in A375 (BRAF V600E), 78.6/35.4 nM in HMV2 (BRAF G469V), 183.0/699.0 nM in SK-MEL-2 (NRAS Q61R), and 53.9/110.0 nM in SK-MEL30 (BRAF D287H and E275K), respectively. KIN-7136 and KIN-8391 synergistically inhibited HMV2, SK-MEL-2, and SK-MEL-30 cells. Pharmacodynamic studies showed that both KIN-7136 and KIN-8391 suppressed phospho-ERK, a downstream component of BRAF-MEK signaling, in A375 xenografts in mice. Daily oral treatment with a combination of KIN-7136 and KIN-8391 was well-tolerated and extended overall survival to 64 days (median survival), compared to the vehicle control and monotherapies with KIN-7136 or KIN-8391 (28, 58, and 46.5, respectively) in the A375 intracranial tumor model. CONCLUSION These preclinical data confirm the synergistic effect of KIN-7136 and KIN-8391 in BRAF-mutant melanoma brain metastases models, supporting further research to advance its clinical translation.

NET-EN treatment leads to delayed HSV-2 infection, enhanced mucin and T cell functions in the female genital tract when compared to DMPA in a preclinical mouse model.

Depot-medroxyprogesterone acetate (DMPA) and Norethisterone Enanthate (NET-EN) are progestin-only injectable contraceptives widely used by women in sub-Sharan Africa, where incidence of HIV-1 and HSV-2 infection remains high. Studies indicate that DMPA usage can increase the risk of HSV-2 infection, but limited data indicate no increased risk with use of NET-EN. We therefore investigated the effects of NET-EN and DMPA on susceptibility to vaginal HSV-2 infection in ovariectomized (OVX) mice and effects on immune responses, particularly in the vaginal tract (VT). OVX mice, when treated with NET-EN and infected intravaginally, had delayed genital pathology, decreased viral shedding, and extended survival compared to DMPA- or untreated OVX mice. CD4+ T cells isolated from VT showed no significant change in frequency with either contraceptive. However, DMPA significantly decreased the total number of VT CD4+ and CD8+ T cells and the number of IFN-γ producing CD4 and CD8 T cells and increased the percentage of CD4 and CD8 T cells producing TNF-α compared to untreated mice. In contrast, NET-EN significantly enhanced percentages of CD8+ T cells compared to DMPA treated mice, and frequencies of IFN-γ+ CD4 and CD8 T cells in the VT compared to untreated mice. Comparative analysis of splenic lymphocytes indicated that DMPA treatment resulted in reduction of CD4+ T cell frequency, but enhanced TNF-α+ CD4 T cells compared to untreated mice. NET-EN enhanced the frequency of CD8 T cells, as well as IFN-γ+ and TNF-α+ CD4, and IFN-γ+ CD8 T cells in the spleen compared to untreated mice. Importantly, we found DMPA treatment that significantly reduced mucin production, whereas NET-EN enhanced expression of cell-associated mucin in VT. High levels of mucin in NET-EN mice were associated with lower levels of HSV-2 virus detected in the vaginal tract. This study provides the first evidence that NET-EN treatment can delay HSV-2 infection compared to DMPA.