Murine Model Research Articles

Ameliorative potential of galangin in murine model of ovalbumin-induced allergic rhinitis: a role of PI3K-PKB pathway.

To examine the potential of galangin in a mouse model of ovalbumin (OVA)-induced allergic rhinitis (AR), as chronic AR, induced by immunoglobulin-E (IgE), leads to histamine release and nasal inflammation, and although galangin exhibits antiasthmatic and anti-inflammatory potential, its effect on AR is yet to be investigated. 126 BALB/c mice. AR induction involved sensitizing female mice with OVA (5%, 500 µL, IP) for 14 days. Post OVA challenge, the mice were divided into 7 groups (n = 18/group), including normal, AR control, montelukast (10 mg/kg), galangin (5, 10, and 20 mg/kg), and per se (galangin [20 mg/kg] treatment. Various outcomes were evaluated, including nasal symptoms, histopathology, biochemistry, and nasal lavage fluid inflammatory cytokines and signaling pathways in nasal mucosal to assess galangin potential in AR. In AR mice, galangin (10 and 20 mg/kg) significantly (P < .05) reduced sneezing, rubbing, and nasal discharge post-OVA challenge. Galangin treatment attenuated (P < .05) elevated serum histamine, β-hexosaminidase, IgE, and Immunoglobulin G1 levels in AR control mice. Additionally, galangin significantly (P < .05) decreased OVA-induced alterations in IL-4, IL-6, IL-13, and interferon-γ levels in nasal lavage fluid compared to AR control mice. Western blot analysis demonstrated that galangin lowered OVA-induced AR by significantly (P < .05) downregulating the phosphorylated protein kinase B and mammalian target of rapamycin-protein expressions while markedly (P < .05) upregulating the glycogen synthase kinase-3β protein expressions in nasal mucosal. Galangin also significantly ameliorated (P < .05) the OVA-induced histological aberrations in the nasal mucosa, reflected by reduced eosinophil infiltration, hyperplasia, and edema. Galangin exhibits antihistaminic and anti-inflammatory effects in AR mice by regulating IgE-mediated histamine and inflammatory release and modulating the phosphatidylinositol 3-kinase/Ak strain transforming/mammalian target of rapamycin pathways.

Description of a Murine Model of Pneumocystis Pneumonia.

Pneumocystis pneumonia is a serious lung infection caused by an original ubiquitous fungus with opportunistic behavior, referred to as Pneumocystis jirovecii. P. jirovecii is the second most common fungal agent among invasive fungal infections after Candida spp. Unfortunately, there is still an inability to culture P. jirovecii in vitro, and so a great impairment to improve knowledge on the pathogenesis of Pneumocystis pneumonia. In this context, animal models have a high value to address complex interplay between Pneumocystis and the components of the host immune system. Here, we propose a protocol for a murine model of Pneumocystis pneumonia. Animals become susceptible to Pneumocystis by acquiring an immunocompromised status induced by iterative administration of steroids within drinking water. Thereafter, the experimental infection is completed by an intranasal challenge with homogenates of mouse lungs containing Pneumocystis murina. The onset of clinical signs occurs within 5weeks following the infectious challenge and immunosuppression can then be withdrawn. At termination, lungs and bronchoalveolar lavage (BAL) fluids from infected mice are analyzed for fungal load (qPCR) and immune response (flow cytometry and biochemical assays). The model is a useful tool in studies focusing on immune responses initiated after the establishment of Pneumocystis pneumonia.

TREM2 protects against inflammation by regulating the release of mito-DAMPs from hepatocytes during liver fibrosis.

Liver fibrosis typically develops as a result of chronic liver injury, which involves inflammatory and regenerative processes. The triggering receptor expressed on myeloid cells 2 (TREM2), predominantly expressing in hepatic non-parenchymal cells, plays a crucial role in regulating the function of macrophages. However, its mechanism in liver fibrosis remains poorly defined. Experimental liver fibrosis models in wild type and TREM2-/- mice, and in vitro studies with AML-12 cells and Raw264.7 cells were conducted. The expression of TREM2 and related molecular mechanism were evaluated by using samples from patients with liver fibrosis. We demonstrated that TREM2 was upregulated in murine model with liver fibrosis. Mice lacking TREM2 exhibited reduced phagocytosis activity in macrophages following carbon tetrachloride (CCl4) intoxication. As a result, there was an increased accumulation of necrotic apoptotic hepatocytes. Additionally, TREM2 knockout aggravated the release of mitochondrial damage-associated molecular patterns (mito-DAMPs) from dead hepatocytes during CCl4 exposure, and further promoted the occurrence of macrophage-mediated M1 polarization. Then, TREM2-/- mice showed more serious fibrosis pathological changes. In vitro, the necrotic apoptosis inhibitor GSK872 effectively alleviated the release of mito-DAMPs in AML-12 cells after CCl4 intoxication, which confirmed that mito-DAMPs originated from dead liver cells. Moreover, direct stimulation of Raw264.7 cells by mito-DAMPs from liver tissue can induce intracellular inflammatory response. More importantly, TREM2 was elevated and inflammatory factors were markedly accumulated surrounding dead cells in the livers of human patients with liver fibrosis. Our study highlights that TREM2 serves as a negative regulator of liver fibrosis, suggesting its potential as a novel therapeutic target.

Functional metabolomics characterizes the contribution of farnesoid X receptor in pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome.

Consumption of herbal products containing pyrrolizidine alkaloids (PAs) is one of the major causes for hepatic sinusoidal obstruction syndrome (HSOS), a deadly liver disease. However, the crucial metabolic variation and biomarkers which can reflect these changes remain amphibious and thus to result in a lack of effective prevention, diagnosis and treatments against this disease. The aim of the study was to determine the impact of HSOS caused by PA exposure, and to translate metabolomics-derived biomarkers to the mechanism. In present study, cholic acid species (namely, cholic acid, taurine conjugated-cholic acid, and glycine conjugated-cholic acid) were identified as the candidate biomarkers (area under the ROC curve 0.968 [95% CI 0.908-0.994], sensitivity 83.87%, specificity 96.55%) for PA-HSOS using two independent cohorts of patients with PA-HSOS. The increased primary bile acid biosynthesis and decreased liver expression of farnesoid X receptor (FXR, which is known to inhibit bile acid biosynthesis in hepatocytes) were highlighted in PA-HSOS patients. Furtherly, a murine PA-HSOS model induced by senecionine (50mg/kg, p.o.), a hepatotoxic PA, showed increased biosynthesis of cholic acid species via inhibition of hepatic FXR-SHP singling and treatment with the FXR agonist obeticholic acid restored the cholic acid species to the normal levels and protected mice from senecionine-induced HSOS. This work elucidates that increased levels of cholic acid species can serve as diagnostic biomarkers in PA-HSOS and targeting FXR may represent a therapeutic strategy for treating PA-HSOS in clinics.

Quantification of Lymphangiogenesis in the Murine Lymphedema Tail Model Using Intravital Microscopy.

Background: Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. It affects an estimated five million Americans. There is no cure for this disease. Assessing lymphatic growth is essential in developing novel therapeutics. Intravital microscopy (IVM) is a powerful imaging tool for investigating various biological processes in live animals. Tissue nanotransfection technology (TNT) facilitates a direct, transcutaneous nonviral vector gene delivery using a chip with nanochannel poration in a rapid (<100 ms) focused electric field. TNT was used in this study to deliver the genetic cargo in the murine tail lymphedema to assess the lymphangiogenesis. The purpose of this study is to experimentally evaluate the applicability of IVM to visualize and quantify lymphatics in the live mice model. Methods and Results: The murine tail model of lymphedema was utilized. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: TNTpCMV6 group receives pCMV6 (expression vector backbone alone) (n = 6); TNTProx1 group receives pCMV6-Prox1 (n = 6). Lymphatic vessels (fluorescein isothiocyanate [FITC]-dextran stained) and lymphatic branch points (indicating lymphangiogenesis) were analyzed with the confocal/multiphoton microscope. The experimental group TNTProx1 exhibited reduced postsurgical tail lymphedema and increased lymphatic distribution compared to TNTpCMV6 group. More lymphatic branching points (>3-fold) were observed at the TNT site in TNTProx1 group. Conclusions: This study demonstrates a novel, powerful imaging tool for investigating lymphatic vessels in live murine tail model of lymphedema. IVM can be utilized for functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy.

Substance specific EEG patterns in mice undergoing slow anesthesia induction

The exact mechanisms and the neural circuits involved in anesthesia induced unconsciousness are still not fully understood. To elucidate them valid animal models are necessary. Since the most commonly used species in neuroscience are mice, we established a murine model for commonly used anesthetics/sedatives and evaluated the epidural electroencephalographic (EEG) patterns during slow anesthesia induction and emergence. Forty-four mice underwent surgery in which we inserted a central venous catheter and implanted nine intracranial electrodes above the prefrontal, motor, sensory, and visual cortex. After at least one week of recovery, mice were anesthetized either by inhalational sevoflurane or intravenous propofol, ketamine, or dexmedetomidine. We evaluated the loss and return of righting reflex (LORR/RORR) and recorded the electrocorticogram. For spectral analysis we focused on the prefrontal and visual cortex. In addition to analyzing the power spectral density at specific time points we evaluated the changes in the spectral power distribution longitudinally. The median time to LORR after start anesthesia ranged from 1080 [1st quartile: 960; 3rd quartile: 1080]s under sevoflurane anesthesia to 1541 [1455; 1890]s with ketamine. Around LORR sevoflurane as well as propofol induced a decrease in the theta/alpha band and an increase in the beta/gamma band. Dexmedetomidine infusion resulted in a shift towards lower frequencies with an increase in the delta range. Ketamine induced stronger activity in the higher frequencies. Our results showed substance-specific changes in EEG patterns during slow anesthesia induction. These patterns were partially identical to previous observations in humans, but also included significant differences, especially in the low frequencies. Our study emphasizes strengths and limitations of murine models in neuroscience and provides an important basis for future studies investigating complex neurophysiological mechanisms.

Are periodontitis and psoriasis associated? A pre-clinical murine model.

To investigate the bidirectional influence between periodontitis and psoriasis, using the respective experimental models of ligature- and imiquimod-induced diseases on murine models. Thirty-two C57/BL6J mice were randomly allocated to four experimental groups: control (P- Pso-), ligature-induced periodontitis (P+ Pso-), imiquimod-induced psoriasis (P- Pso+) and periodontitis and psoriasis (P+ Pso+). Samples (maxilla, dorsal skin and blood) were harvested immediately after death. Measures of periodontitis (distance between the cemento-enamel junction and alveolar bone crest [CEJ-ABC] and the number of osteoclasts) and psoriasis (epidermal thickness and infiltrate cell [/0.03mm2]) severity as well as systemic inflammation (IL-6, IL-17A, TNF-α) were collected. The P+ Pso+ group exhibited the most severe experimental periodontitis and psoriasis, with the highest values of CEJ-ABC, number of osteoclasts, epidermal thickness and infiltrate cells in the dorsal skin, as well as the highest blood cytokine concentration. The P+ Pso- group presented with higher cell infiltrate (/0.03mm2) compared to the control group (p <.05), while the P- Pso+ group showed substantially higher alveolar bone loss (CEJ-ABC) than the control group (p <.05). Experimental periodontitis may initiate and maintain psoriasiform skin inflammation and, vice versa, experimental psoriasis may contribute to the onset of periodontitis. In a combined model of the diseases, we propose a bidirectional association between periodontitis and psoriasis via systemic inflammation.

Improving Cutaneous Wound Healing in Diabetic Mice Using Naturally Derived Tissue-Engineered Biological Dressings Produced under Serum-Free Conditions

Long-term diabetes often leads to chronic wounds refractory to treatment. Cell-based therapies are actively investigated to enhance cutaneous healing. Various cell types are available to produce biological dressings, such as adipose-derived stem/stromal cells (ASCs), an attractive cell source considering their abundancy, accessibility, and therapeutic secretome. In this study, we produced human ASC-based dressings under a serum-free culture system using the self-assembly approach of tissue engineering. The dressings were applied every 4 days to full-thickness 8-mm splinted skin wounds created on the back of polygenic diabetic NONcNZO10/LtJ mice and streptozotocin-induced diabetic K14-H2B-GFP mice. Global wound closure kinetics evaluated macroscopically showed accelerated wound closure in both murine models, especially for NONcNZO10/LtJ; the treated group reaching 98.7% ± 2.3% global closure compared to 76.4% ± 11.8% for the untreated group on day 20 (p=0.0002). Histological analyses revealed that treated wounds exhibited healed skin of better quality with a well-differentiated epidermis and a more organized, homogeneous, and 1.6-fold thicker granulation tissue. Neovascularization, assessed by CD31 labeling, was 2.5-fold higher for the NONcNZO10/LtJ treated wounds. We thus describe the beneficial impact on wound healing of biologically active ASC-based dressings produced under an entirely serum-free production system facilitating clinical translation.

A Systematic Review and Meta-Analysis of microRNA Profiling Studies in Chronic Kidney Diseases

Chronic kidney disease (CKD) represents an increasing health burden. Evidence suggests the importance of miRNA in diagnosing CKD, yet the reports are inconsistent. This study aimed to determine novel miRNA biomarkers and potential therapeutic targets from hypothesis-free miRNA profiling studies in human and murine CKDs. Comprehensive literature searches were conducted on five databases. Subgroup analyses of kidney diseases, sample types, disease stages, and species were conducted. A total of 38 human and 12 murine eligible studies were analyzed using Robust Rank Aggregation (RRA) and vote-counting analyses. Gene set enrichment analyses of miRNA signatures in each kidney disease were conducted using DIANA-miRPath v4.0 and MIENTURNET. As a result, top target genes, Gene Ontology terms, the interaction network between miRNA and target genes, and molecular pathways in each kidney disease were identified. According to vote-counting analysis, 145 miRNAs were dysregulated in human kidney diseases, and 32 were dysregulated in murine CKD models. By RRA, miR-26a-5p was significantly reduced in the kidney tissue of Lupus nephritis (LN), while miR-107 was decreased in LN patients’ blood samples. In both species, epithelial-mesenchymal transition, Notch, mTOR signaling, apoptosis, G2/M checkpoint, and hypoxia were the most enriched pathways. These miRNA signatures and their target genes must be validated in large patient cohort studies.

Time-course RNAseq data of murine AB1 mesothelioma and Renca renal cancer following immune checkpoint therapy

Time-critical transcriptional events in the immune microenvironment are important for response to immune checkpoint blockade (ICB), yet these events are difficult to characterise and remain incompletely understood. Here, we present whole tumor RNA sequencing data in the context of treatment with ICB in murine models of AB1 mesothelioma and Renca renal cell cancer. We sequenced 144 bulk RNAseq samples from these two cancer types across 4 time points prior and after treatment with ICB. We also performed single-cell sequencing on 12 samples of AB1 and Renca tumors an hour before ICB administration. Our samples were equally distributed between responders and non-responders to treatment. Additionally, we sequenced AB1-HA mesothelioma tumors treated with two sample dissociation protocols to assess the impact of these protocols on the quality transcriptional information in our samples. These datasets provide time-course information to transcriptionally characterize the ICB response and provide detailed information at the single-cell level of the early tumor microenvironment prior to ICB therapy.

A murine model of post-acute neurological sequelae following SARS-CoV-2 variant infection

Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. In the brain, no detectable viral RNA and minimal residential immune cell activation was observed in the surviving mice post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and gene expression levels associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent systemic T helper 1 prone cellular immune responses and strong sera neutralizing antibodies against Delta and Omicron variants months post-acute infection. Overall, our findings suggest that infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long-COVID patients and provides new insights into the role of systemic and brain residential immune factors in PASC pathogenesis.

Conventional type 1 dendritic cells are essential for the development of primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a progressive-cholestatic autoimmune liver disease. Dendritic cells (DC) are professional antigen-presenting cells and their prominent presence around damaged bile ducts of PBC patients are documented. cDC1 is a rare subset of DC known for its cross-presentation abilities and interleukin 12 production. Our aim was to assess the role of cDC1 in the pathogenesis of PBC. We utilized an inducible murine model of PBC and took advantage of the DC reporter mice Zbtb46gfp and the Batf3-/- mice that specifically lack the cDC1 subset. cDC1 cells were sorted from blood of PBC patients and healthy individuals and subjected to Bulk-MARS-seq transcriptome analysis. Histopathology assessment demonstrated peri-portal inflammation in wild type (WT) mice, whereas only minor abnormalities were observed in Batf3-/- mice. Flow cytometry analysis revealed a two-fold reduction in hepatic CD8/CD4 T cells ratio in Batf3-/- mice, suggesting reduced intrahepatic CD8 T cells expansion. Histological evidence of portal fibrosis was detected only in the WT but not in Batf3-/- mice. This finding was supported by decreased expression levels of pro-fibrotic genes in the livers of Batf3-/- mice. Transcriptome analysis of human cDC1, revealed 78 differentially expressed genes between PBC patients and controls. Genes related to antigen presentation, TNF and IFN signalling and mitochondrial dysfunction were significantly increased in cDC1 isolated from PBC patients. Our data illustrated the contribution the cDC1 subset in the pathogenesis of PBC and provides a novel direction for immune based cell-specific targeted therapeutic approach in PBC.

Beneficial Effects of Oral Carbon Monoxide on Doxorubicin-Induced Cardiotoxicity.

Doxorubicin and other anthracyclines are crucial cancer treatment drugs. However, they are associated with significant cardiotoxicity, severely affecting patient care and limiting dosage and usage. Previous studies have shown that low carbon monoxide (CO) concentrations protect against doxorubicin toxicity. However, traditional methods of CO delivery pose complex challenges for daily administration, such as dosing and toxicity. To address these challenges, we developed a novel oral liquid drug product containing CO (HBI-002) that can be easily self-administered by patients with cancer undergoing doxorubicin treatment, resulting in CO being delivered through the upper gastrointestinal tract. HBI-002 was tested in a murine model of doxorubicin cardiotoxicity in the presence and absence of lung or breast cancer. The mice received HBI-002 twice daily before doxorubicin administration and experienced increased carboxyhemoglobin levels from a baseline of ≈1% to 7%. Heart tissue from mice treated with HBI-002 had a 6.3-fold increase in CO concentrations and higher expression of the cytoprotective enzyme heme oxygenase-1 compared with placebo control. In both acute and chronic doxorubicin toxicity scenarios, HBI-002 protected the heart from cardiotoxic effects, including limiting tissue damage and cardiac dysfunction and improving survival. In addition, HBI-002 did not compromise the efficacy of doxorubicin in reducing tumor volume, but rather enhanced the sensitivity of breast 4T1 cancer cells to doxorubicin while simultaneously protecting cardiac function. These findings strongly support using HBI-002 as a cardioprotective agent that maintains the therapeutic benefits of doxorubicin cancer treatment while mitigating cardiac damage.

Sodium oligomannate activates the enteroendocrine-vagal afferent pathways in APP/PS1 mice.

Enteroendocrine cells (EECs) and vagal afferent neurons constitute functional sensory units of the gut, which have been implicated in bottom-up modulation of brain functions. Sodium oligomannate (GV-971) has been shown to improve cognitive functions in murine models of Alzheimer's disease (AD) and recently approved for the treatment of AD patients in China. In this study, we explored whether activation of the EECs-vagal afferent pathways was involved in the therapeutic effects of GV-971. We found that an enteroendocrine cell line RIN-14B displayed spontaneous calcium oscillations due to TRPA1-mediated calcium entry; perfusion of GV-971 (50, 100 mg/L) concentration-dependently enhanced the calcium oscillations in EECs. In ex vivo murine jejunum preparation, intraluminal infusion of GV-971 (500 mg/L) significantly increased the spontaneous and distension-induced discharge rate of the vagal afferent nerves. In wild-type mice, administration of GV-971 (100 mg· kg-1 ·d-1, i.g. for 7 days) significantly elevated serum serotonin and CCK levels and increased jejunal afferent nerve activity. In 7-month-old APP/PS1 mice, administration of GV-971 for 12 weeks significantly increased jejunal afferent nerve activity and improved the cognitive deficits in behavioral tests. Sweet taste receptor inhibitor Lactisole (0.5 mM) and the TRPA1 channel blocker HC-030031 (10 µM) negated the effects of GV-971 on calcium oscillations in RIN-14B cells as well as on jejunal afferent nerve activity. In APP/PS1 mice, co-administration of Lactisole (30 mg ·kg-1 ·d-1, i.g. for 12 weeks) attenuated the effects of GV-971 on serum serotonin and CCK levels, vagal afferent firing, and cognitive behaviors. We conclude that GV-971 activates sweet taste receptors and TRPA1, either directly or indirectly, to enhance calcium entry in enteroendocrine cells, resulting in increased CCK and 5-HT release and consequent increase of vagal afferent activity. GV-971 might activate the EECs-vagal afferent pathways to modulate cognitive functions.

Abstract PO5-27-02: A Tumor Emboli Platform for Morphometric Analysis, Drug screening and for Intravital Optical Imaging in a Murine Model to Simulate Clinicopathological Features of Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is distinguished by the presence of tumor emboli, clusters of tumor cells, found within breast parenchyma and dermal lymphatic vessels. These emboli serve as a crucial clinicopathological hallmark, believed to underlie the characteristic diffuse growth pattern and aggressive progression observed in IBC patients, with nearly 30% of patients presenting with metastatic disease at diagnosis. However, the rarity of this cancer and the absence of a solid tumor mass pose challenges in obtaining biospecimens, particularly those with evidence of tumor emboli. To address this pressing need for preclinical models that replicate this distinctive tumor growth pattern, we describe a tumor emboli culture platform that facilitates live imaging, enables compound screening by assessing various phenotypes of the emboli, and permits evaluation of emboli in an in vivo murine model. Methods: The tumor emboli culture system was evaluated for physiological measurements corresponding to dermal lymphatic vessels and subsequently employed to generate tumor emboli from diverse patient-derived cell lines and PDX. Confocal microscopy was employed to characterize cellular information across multiple slices of the tumor emboli. This provided a comprehensive, multi-parametric analysis of single-cell and spheroid phenotypes, including measurements of number, size, shape, and viability of the organoids. Furthermore, we developed imaging algorithms to evaluate phenotypic changes in tumor emboli in response to anti-cancer drug treatment. We also optimized a technique that allows for in vivo tumor emboli implantation including in a transgenic mice with red fluorescent lymphatic vasculature that were surgically implanted with a dorsal skin window chamber. This allowed for intravital optical imaging of the local tumor microenvironment and adjacent skin. Results: Our culture platform enables live imaging of the tumor emboli generated over a span of 7-10 days, maintaining key parameters including kinematic and dynamic viscosity, density along with sheer wall stress within the reported physiological range for lymphatics. Through higher resolution confocal image acquisition and multi-parametric analysis, we conducted particle counting and classification, providing statistically significant insights into the integrity of tumor emboli over time. Additionally, the developed algorithms allowed for quantitative analysis of images from tumor emboli cultures subjected to anti-cancer drugs, revealing disparities in drug efficacy on tumor emboli integrity and dispersion including inter-cluster space within the emboli. Intravital optical imaging, coupled with quantitative analysis of dispersion characteristics of the tumor emboli implanted in the murine model over 5-6 days, demonstrated a diffuse spread reminiscent of observations in IBC patients. Furthermore, immunohistochemistry staining showed marked Ki67, CD45, F4-80 revealing a proliferative tumor microenvironment with high levels of macrophage infiltration. Conclusions: As of now specific targeted therapies for IBC patients remain elusive. Our culture platform for tumor emboli, when combined with advanced imaging techniques both in vitro and in vivo and traditional approaches like immunohistochemistry and genomic evaluation, forms a powerful toolkit for assessing the phenotype and dispersion characteristics of this unique pathological feature in IBC. This preclinical model offers crucial insights, potentially paving the way for precision medicine strategies in the treatment of this understudied cancer. Funding in part from American Cancer Society Mission Boost MBG-20-141-01-MBG grant (GRD), Department of Defense W81XWH-20-1-0153 (GRD), Duke Consortium for Inflammatory Breast Cancer Education (AB, CW, RE). Citation Format: Dorababu Sannareddy, Theresa Charity, Alexandra Bennion, Caroline Way, Edwin Yu, Ralph Erdmann, Gregory M Palmer, Gayathri R Devi. A Tumor Emboli Platform for Morphometric Analysis, Drug screening and for Intravital Optical Imaging in a Murine Model to Simulate Clinicopathological Features of Inflammatory Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-02.

Abstract PS18-09: The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment

Abstract Background. Despite a recent convergence of breast cancer (BC) incidence rates between African American (AA) and European American (EA) women, AA women continue to have ~40% higher mortality rates. BC is a heterogeneous collection of diseases, where African ancestry women are disproportionately burdened with the most aggressive subtype of BC, triple negative BC (TNBC). We have previously reported that the African ancestry-specific Duffy-null allele (FY-) is a risk factor for TNBC diagnoses. FY- is a promoter variant that removes expression of the Duffy Antigen Receptor for Chemokines (DARC/ACKR1), from red blood cells (RBCs). DARC serves as a portal of entry for Plasmodium vivax malaria parasite, and FY- confers immunity, leading to fixation of FY- among Sub-Saharan African populations. DARC is an atypical chemokine receptor that functions to modulate chemokine levels in circulation and tissues to aid immune cell recruitment. DARC is also expressed on breast tumor epithelial cells, however the significance of DARC in the TNBC tumor microenvironment (TME) has not fully been explored. Leveraging our African ancestry enriched International Study for the Center of Breast Cancer Subtypes cohort and in vivo murine models, we employed multi-omics approaches to explore the function of FY- and DARC expression across cell types in the TNBC TME. Genomics. Using transcriptomic data from the TCGA BC cohort, we have previously reported that DARC expression is highly correlated with deconvoluted immune cell abundance using CIBERSORT. To build upon this finding among TNBC patients, we have analyzed RNAseq data from two cohorts of African ancestry women to explore correlation of DARC expression status and immune cell abundance. Among TNBC cases, we report that higher DARC expression was significantly associated with increased immune cell abundance, with increases in B cell, T cell and monocyte populations. Using whole genome sequencing data across 86 African ancestry cases, we are currently investigating germline FY- status with mutational signatures. Circulating biomarkers. Patient plasma was collected prior to surgery, where Luminex multiplex assays were performed to quantify levels of &amp;gt;40 circulating chemokines and cytokines. FY- status has been determined for ~550 patients using single-plex or multiplex PCR genotyping methods from germline saliva DNA. Our univariate analyses show several circulating chemokines, including CCL2, CXCL2, CXCL6 and CXCL11, have significant differences between FY- and non-FY- BC patients. Curation of clinicopathological variables for multivariate analysis is ongoing. Murine models. We crossed C3(1)Tag BC transgenic mice with a DARC knock-out (KO) line to generate mice lacking DARC expression that spontaneously develop tumors in the mammary fat pads. The C3(1)Tag model is reported to have similar gene expression profiles to basal-like TNBC tumors. Tumors were collected at pre-determined time points of 25 and 30 weeks, where DARC KO mice had increased tumor size and tumor burden at 30 weeks. Initial fluorescent staining revealed an increase in immune cell infiltration in the TME of DARC expressing mice compared to DARC KO mice. To further explore this, we employed multiplex staining across a representative cohort of DARC expressing and KO mice at 25 and 30 weeks to capture the specific immune cell populations infiltrating the TME. Conclusions. TNBC tumors have worse prognosis due to lack of targeted therapeutic options. Increased immunogenicity of TNBC tumors, especially among African ancestry women may be driven in part by DARC expression. Characterization of FY- status, in coordination with DARC among other cell types in the TNBC TME could present new opportunities for biomarker and/or therapeutic development to improve prognosis for these underserved populations. Citation Format: Rachel Martini, Stevens Patino, Emma Guyonnet, Brian Stonaker, Isra Elhussein, Julie Sahler, Avery August, Nancy Manley, Rick Kittles, Clayton Yates, Lisa Newman, Melissa Davis. The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS18-09.

A novel inhibitor of the mitochondrial respiratory complex I with uncoupling properties exerts potent antitumor activity

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy.MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.

Abstract PO1-18-04: Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968)

Abstract Background: Higher levels of stromal tumor-infiltrating lymphocytes (sTILs) in breast cancers are associated with increased likelihood of pathologic complete response (pCR) to chemotherapy and improved outcomes. DNA methyltransferase inhibitors (DNMTi) can augment immune responses to cancers by upregulating tumor antigen and MHC expression, decreasing numbers and activity of myeloid derived suppressor cells (MDSC), and increasing responsiveness of T lymphocytes. We have shown that the DNMTi decitabine augments the effectiveness of immunotherapy against murine triple negative breast cancer (TNBC) using murine 4T1 and E0771 mammary carcinoma models. Methods: In a single-arm phase 2 study, patients with HER2-negative breast cancer who were candidates for neoadjuvant chemotherapy (NCT) received decitabine (15 mg/m2 × 4 doses over 5 days) followed by 2 doses of pembrolizumab (pembro) (200 mg, 2 weeks apart) – collectively, window immunotherapy – prior to starting NCT. Two research biopsies were obtained: 1 prior to the window immunotherapy and 1 afterwards, prior to starting NCT. Biopsies were analyzed using established procedures to quantify sTILs and PD-L1 expression. Patients proceeded to NCT and tumor resection per standard of care and were followed up for immune-related adverse events (irAEs) and response. The primary endpoint was change in sTILs. Key secondary endpoints were occurrence of irAEs and pCR following neoadjuvant treatment. After the study was opened, reported results of a phase 3 trial (KEYNOTE 522) led to allowing patients with TNBC to receive additional pembro (200 mg q3w) concurrently with standard NCT and adjuvantly following resection (Cohort A2). Results: 46 patients (median age 54.5 yrs, range 28-72; 71.7% White, 28.3% Black; 100% female) were enrolled and treated on study. 21 patients had TNBC and did not receive neoadjuvant pembro concurrently with NCT nor adjuvant pembro (Cohort A), 7 patients had TNBC and did receive neoadjuvant and/or adjuvant pembro (Cohort A2), and 18 patients were ER+ or PR+ and received neither concurrent nor adjuvant pembro (Cohort B). Blood samples collected after decitabine administration before the first pembro dose showed a 59% decrease (P&amp;lt; 0.01) in monocytic MDSCs compared to baseline; decrease in granulocytic MDSCs was not statistically significant. 37 patients had paired biopsies adequate for sTIL evaluation with a mean change from 23.4% to 30.3% (absolute change 6.9%, P&amp;lt; 0.001). Cohorts A/A2 experienced an absolute sTIL increase of 7.4% (P&amp;lt; 0.01); Cohort B experienced absolute sTIL increase of 6.1% (P=0.01). PD-L1 expression in tumors (H-score using MoAb 22C3 clone) increased by 43% (P&amp;lt; 0.01) across all cohorts. 16 of the 39 patients (41.0%) who proceeded to resection achieved pCR (n=12 of 27 [44.4%] in Cohorts A/A2 and n=4 of 13 [30.8%] in Cohort B). The most frequently reported irAEs were adrenal insufficiency (n=5, 10.9%), maculopapular rash (n=3, 6.5%), and hypothyroidism (n=3, 6.5%). Conclusions: Treatment in the pre-neoadjuvant window with decitabine and pembro could potentially sensitize breast cancers to standard NCT by recruitment of TILs to the tumor tissue. The treatment was well -tolerated at the tested doses. Funding: financial support and drug (pembrolizumab) were provided by Merck Citation Format: Harry Bear, Xiaoyan Deng, Dipankar Bandyopadhyay, Michael Idowu, Maciej Kmieciak, Monique Williams, Giovanni Archer, Lindsey Gwaltney, Patrick Dillon, Daniel Flora, Daniel Stover, Andrew Poklepovic, Mary Hackney, Masey Ross, Hetal Vachhani, Raphael Louie, Kandace McGuire, Amelia Grover, Tasnim Rahman, Amber Hendrix. Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-18-04.

Abstract ED03-01: Breaking the Obesity-Breast Cancer Link: Comparing Diet, Drug and Surgical Approaches

Abstract The prevalence of obesity, an established risk and progression factor for at least 15 types of cancer (including breast cancer), continues to rise in the US and many other countries. Lifestyle approaches to weight loss have proven challenging for most people to maintain, and bariatric surgery, while effective, is expensive and has numerous adverse effects. The recent availability of highly effective incretin therapies for weight loss raises several key questions that we will address, including: i) do these agents drive weight loss in obese female mice?; ii) do they reverse obesity-induced metabolic dysregulation?; iii) can they reverse obesity-driven cancer?; iv) how heterogeneous is the response to these agents? We will report our study of the effects of extended (13 weeks) tirzepatide (TZP, a dual GLP1R/GIPR agonist) treatment in obese female mice on weight loss, metabolic hormones, and mammary tumor growth. We found that—similar to reports in the literature using male mice—obese female mice lost &amp;gt;20% body weight by 4 weeks of TZP treatment. Moreover, TZP-induced weight loss was maintained through 13 weeks of treatment by progressively escalating the dose up to 40 nmol/kg. Congruent with reports in male rodents and humans, we found the majority of weight loss in female mice was attributed to loss of adipose tissue, rather than lean mass. The effects of TZP on body weight and composition were associated with reversal of obesity-induced changes in glycemic control and circulating levels of metabolic hormones, particularly leptin, insulin, and IGF-1. TZP also significantly reversed the protumor effects of obesity in our murine E0771 orthotopic transplant model of TNBC.Furthermore, daily 30% calorie restriction, which we previously reported has strong weight loss-inducing, metabolic reprogramming and anticancer effects in multiple mouse models of breast and other cancers, reversed body weight, adiposity, and TNBC tumor growth and altered metabolic hormones to a greater extent than all groups studied, including the never-obese control mice and TZP-treated mice. Citation Format: S. Hursting. Breaking the Obesity-Breast Cancer Link: Comparing Diet, Drug and Surgical Approaches [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr ED03-01.

Abstract PO5-24-01: Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models

Abstract Despite improvement of outcomes for HR+, HER2- metastatic breast cancer by cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors, safety concerns and disease progression raise a critical need to identify novel approaches. Narazaciclib (ON123300), a novel CDK4/6i, designed to enhance efficacy and safety by its multi-targeted kinase inhibitor activity. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; studying different administration regimens and in endometrial cancer in combination with letrozole (NCT05705505). Our study explored the activity of narazaciclib and its metabolite ON1232580 in comparisons to the FDA approved CDK4/6i and identify additional targets engaged by narazaciclib. To identify direct and secondary targets engaged by narazaciclib and palbociclib, proteome wide Cellular Thermal Shift Assay (CETSA) and Integrative Inferred Kinase Activity (INKA) analysis were performed. Both CETSA and INKA analysis identified additional targets engaged by narazaciclib compared to palbociclib in both MDA-MB-231 lysates and intact cells such as BUB1, CHEK1, AURKA, GSK3α and GSK3β. In TNBC patients with BUB1 overexpression, bioinformatics analysis indicates a low survival correlation. Molecular docking data showed a higher affinity of narazaciclib with BUB1 compared to palbociclib and abemaciclib. The differential activity of narazaciclib was explored by using cohorts of human and mouse breast cancer cell lines and cell based assays. Comparison of narazaciclib’s in vitro IC50 profile to other CDK4/6i was studied against a panel of 370 kinases (HotSpot) and Kd values were determined by KINOMEscan. Intracellular IC50 kinase values were determined by NanoBret technology. Narazaciclib and abemaciclib were found to be the most promiscuous in vitro kinase inhibitors and ribociclib the most specific. Abemaciclib and narazaciclib had similar profiles against the CDK family members. Kd values of CDK4/cyclinD1 binding show similar trends; abemaciclib (0.08 nM), narazaciclib (0.18 nM), palbociclib (0.75 nM) and ribociclib (1.3 nM). Although narazaciclib displayed nM IC50 values in the in vitro assays against many CDKs, narazaciclib was very specific in cellular kinase assays with highest activity against CDK4/6, CSF1R and NUAK1. A stronger inhibition of cell proliferation, as well as induction of apoptosis and senescence were detected in narazaciclib and its metabolite ON1232580 treated PYMT cells, a murine mammary carcinoma model which recapitulates luminal BC subtype, compared to the other CDK4/6i. Both narazaciclib and ON1232580 enhanced CCL5 and CXCL10 mRNA levels, while a higher reduction of PD-L1 and pRb protein levels and a promoting effect on the H2D1 and B2M mRNA levels was shown in narazaciclib and its metabolite treated PYMT cells. Narazaciclib showed significant synergy with anti-PD1 in the EMT-6 syngeneic breast cancer model. A differential effect of narazaciclib on the expression levels of 62 cytokines was detected compared to abemaciclib and palbociclib. Lastly, inhibition of autophagy sensitizes cancer cells to both ON123300 and ON1232580, and induces irreversible cell proliferation inhibition, providing a novel therapeutic approach. Our findings identify important differences generated from assay models studying kinase inhibition, binding and pathway engagement. Understanding the role of the differential targets engaged by narazaciclib, the potential enhanced antitumor immunity and the sensitization by autophagy inhibitors to cell death, will guide future clinical studies. Citation Format: Petros Kechagioglou, Debomita Chakraborty, Camille Dupont, Hajime Yurugi, Ute Distler, Stefan Tenzer, Alexey Chernobrovkin, Kristina Riegel, Julianne Mooz, Mahil Lamber, Volker Dötsch, Stephen Cosenza, Steven Fruchtman, Krishnaraj Rajalingam. Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-01.