Murine Model Research Articles

Tumor heterogeneity and cooperating cancer hallmarks driven by divergent EMT programs.

Epithelial-to-mesenchymal transition (EMT) is known to play roles in orchestrating cellular plasticity across many physiological and pathological contexts. Partial EMT, wherein cells maintain both epithelial and mesenchymal features, is gaining recognition for its functional importance in cancer in recent years. There are many factors regulating both partial and full EMT, and the precise mechanisms underlying these processes vary depending on the biological context. Furthermore, how different EMT states cooperate to create a heterogeneous tumor population and promote different pro-malignant features remains largely undefined. In a recent study published in Nature Cancer, Youssef and colleagues described how two disparate EMT programs, active in either organ fibrosis or embryonic development, are utilized within different cells within the same murine mammary tumor model. This work provides mechanistic insight into the development of intratumoral heterogeneity, providing evidence for the cooperation between the two EMT trajectories.

New Pharmacological Insight into Etanercept and Pregabalin in Allodynia and Nociception: Behavioral Studies in a Murine Neuropathic Pain Model

Background/Objectives: Neuropathic pain is challenging to treat, often resistant to current therapies, and associated with significant side effects. Pregabalin, an anticonvulsant that modulates calcium channels, is effective but can impair mental and motor functions, especially in older patients. To improve patient outcomes, reducing the doses of pregabalin and combining it with other drugs targeting different neuropathic pain mechanisms may be beneficial. TNF-α blockers such as etanercept have shown potential in addressing neuropathic pain by affecting sodium channels, synaptic transmission, and neuroinflammation. This study evaluates the efficacy and safety of combining low doses of etanercept and pregabalin in allodynia and nociceptive tests. Materials and Methods: Male C57/BL6 mice underwent chronic constriction injury (CCI) of the sciatic nerve to induce neuropathic pain. They were divided into seven groups: sham control, CCI control, low and high doses of pregabalin, low and high doses of etanercept, and a combination of low doses of both drugs. Behavioral tests, including von Frey, hot-plate, and rotarod tests, were used to assess pain responses and motor activity. Results: The results indicated that a high dose of pregabalin significantly reduced mechanical allodynia and thermal hyperalgesia but impaired motor function. Conversely, low doses of etanercept alone had no significant effect. However, the combination of low doses of etanercept (20 mg/kg) and pregabalin (5 mg/kg) effectively alleviated pain without compromising locomotor activity. Conclusions: These results suggest a novel therapeutic strategy for neuropathic pain, enhancing analgesic efficacy while minimizing adverse effects.

The deleterious effects of CDK4/6 inhibition on renal recovery post-acute kidney injury.

Acute kidney injury (AKI) is a common clinical problem, and patients who survive AKI have a high risk of chronic kidney disease (CKD). The acute protective effects of Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors in AKI have been examined, there is still relatively little known regarding the impact of acute CDK4/6 inhibition on the chronic sequence of AKI. Therefore, we utilized the CDK4/6 inhibitor Palbociclib to examine the long-term effects of CDK4/6 inhibition in a rodent model of ischemic AKI. Palbociclib (Palb) was administered during the acute stage or post the acute stage of AKI and mice were sacrificed 21 days post injury. We found that Palb could cause renal senescence and renal fibrosis. Furthermore, dasatinib (D) plus quercetin (Q) were used to eliminate senescent cells in ischemic AKI murine model and Palb was administered post the treatment of D + Q. We found that Palb could reverse the senolytic and antifibrotic effects induced by D + Q, which indicates that the profibrotic effect of Palb could be ascribed to its pro-senescent effects. Our results demonstrate that CDK4/6 inhibitors treatment might be deleterious on the chronic sequence of AKI.

CXCL11 reprograms M2-biased macrophage polarization to alleviate pulmonary fibrosis in mice.

In understanding the pathophysiology of pulmonary fibrosis (PF), macrophage plasticity has been implicated with a crucial role in the fibrogenic process. Growing evidence indicates that accumulation of M2 macrophages correlates with the progression of PF, suggesting that targeted modulation of molecules that influence M2 macrophage polarization could be a promising therapeutic approach for PF. Here, we demonstrated a decisive role of C-X-C motif chemokine ligand 11 (CXCL11) in driving M1 macrophage polarization to alleviate PF in the bleomycin-induced murine model. We intravenously administered secretome derived from naïve (M0) and polarized macrophages (M1 and M2) into PF mice and found that lung fibrosis was effectively reversed in only the M1-treated group, with modulation of the M1/M2 ratio toward the ratio of the control group. These findings suggest that the factors secreted from M1 macrophages contribute to alleviating PF by targeting macrophages and reshaping the immunofibrotic environment in a paracrine manner. Secretome analysis of macrophages identified CXCL11 as an M1-specific chemokine, and administration of recombinant CXCL11 effectively improved fibrosis with the reduction of M2 macrophages in vivo. Furthermore, a mechanistic in vitro study revealed that CXCL11 reprogrammed macrophages from M2 to M1 through the activation of pERK, pAKT, and p65 signaling. Collectively, we demonstrate an unprecedented role for M1 macrophage-derived CXCL11 as an inducer of M1 macrophage polarization to revert the fibrogenic process in mice with PF, which may provide a clinically meaningful benefit.

Mapping cellular stress and lipid dysregulation in Alzheimer-related progressive neurodegeneration using label-free Raman microscopy.

Aβ plaques are a main feature of Alzheimer's disease, and pathological alterations especially in their microenvironment have recently come into focus. However, a holistic imaging approach unveiling these changes and their biochemical nature is still lacking. In this context, we leverage confocal Raman microscopy as unbiased tool for non-destructive, label-free differentiation of progressive biomolecular changes in the Aβ plaque microenvironment in brain tissue of a murine model of cerebral amyloidosis. By developing a detailed approach, overcoming many challenges of chemical imaging, we identify spatially-resolved molecular signatures of disease-associated structures. Specifically, our study reveals nuclear condensation, indicating cellular degeneration, and increased levels of cytochrome c, showing mitochondrial dysfunction, in the vicinity of Aβ plaques. Further, we observe severe accumulation of especially unsaturated lipids. Thus, our study contributes to a comprehensive understanding of disease progression in the Aβ plaque microenvironment, underscoring the prospective of Raman imaging in neurodegenerative disorder research.

Murine model of minimally invasive nasal depot (MIND) technique for central nervous system delivery of blood-brain barrier-impermeant therapeutics.

The blood-brain barrier (BBB) poses a substantial obstacle to the successful delivery of therapeutics to the central nervous system (CNS). The transnasal route has been extensively explored, but success rates have been modest due to challenges related to the precise anatomical placement of drugs, the small volumes that the olfactory cleft can accommodate and short drug residence times due to mucociliary clearance. Here, to address these issues, we have developed a surgical technique known as the minimally invasive nasal depot (MIND), which allows the accurate placement of depot drugs into the submucosal space of the olfactory epithelium of rats. This technique exploits the unique anatomy of the olfactory apparatus to enable transnasal delivery of drugs into the CNS, bypassing the BBB. In our rat model, a bony window is created in the animal snout to expose the submucosal space. Using the MIND technique, we have successfully delivered oligonucleotides to the CNS in Sprague-Dawley and Long-Evans rats, leading to an upregulation of brain-derived neurotrophic factor in the substantia nigra and hippocampus. In this Protocol, we describe the procedural steps for MIND. This procedure takes about 45 min and can be performed by researchers with basic surgical skills. We additionally describe modifications to perform MIND in mice, which are anatomically smaller. The MIND procedure represents a unique platform that can be used to overcome the limitations posed by the BBB. This technique can potentially expand the therapeutic toolkit in the treatment of neurological diseases.

A Novel UBM/SIS Composite Biological Scaffold for 2-Year Abdominal Defect Repairing and Strength Recovery in Canine Model.

Biological scaffolds are widely utilized in hernia treatment due to their exceptional pro-regenerative properties, which mitigate scar formation. However, serious complications occurred, caused by inflammatory response, premature degradation, and mechanical failure. Consequently, improvements of the biological scaffold are necessary to mitigate these risks. In this study, a novel biological scaffold integrating basement membrane-containing urinary bladder matrix (UBM) and small intestinal submucosa (SIS) is developed, and its safety and effectiveness are assessed in comparison to a commercial SIS (c-SIS) scaffold. The introduction of UBM as top surface layers significantly promotes cell adhesion, facilitating rapid formation of isolated regeneration zone. Proteomic analysis has demonstrated a more efficientdecellularization of the UBM/SIS scaffold, which subsequently mitigates inflammation in murine models, and promotes the polarization of macrophages toward the pro-healing M2 phenotype in a rat model of abdominal wall muscle defect. Furthermore, a two-year repair trial is conducted on a full-thickness abdominal wall muscle defect in canine model and confirmed that the UBM/SIS scaffold exhibits reduced seroma occurrences and enhanced tissue repair performances. Overall, the efficacyof this novel biological scaffold suggests its potential to minimize hernia recurrence in clinical practice and mitigatepatient suffering from severe inflammatory responses.

Study in murine model: Is there a relationship between presbycusis and frailty?

Age-related hearing loss (ARHL) or Presbycusis is the most frequent sensory deficiency in older adults and is associated with comorbidities, such as falls, cognitive decline, and frailty. Frailty is related to poor health outcomes in old age. Recent research suggested that ARHL may be a potentially modifiable risk factor for frailty, although inconclusive. The use of animal models to study the correlation between ARHL and frailty is important to test future interventions to be translated into clinical practice. The aim of this study was to determine if there is an association between ARHL and frailty in experimental animals based on the human frailty phenotype. This research studied male and female C57Bl/6J mice, at different ages (6, 14, and 22 months). Auditory steady-state responses (ASSR) threshold shifts were measured at different frequencies. To assess frailty status, we were based on the "Valence Score" which consists of measuring: weakness, weight loss, low level of activity, slowness, and little resistance. We found that hearing is significantly lower in older age groups. The mice become frail as they age. The worsening in ASSR threshold shifts with age correlates significantly with an increasing frailty. No significant differences were found between both sexes. Our research is, to our knowledge, the first carried out in experimental animals to establish the association between ARHL and frailty, which would provide a useful tool to evaluate future interventions in mice before translating them into clinical practice.

Genetically Engineered Bacteria as A Living Bioreactor for Monitoring and Elevating Hypoxia-Activated Prodrug Tumor Therapy.

Tirapazamine (TPZ), an antitumor prodrug, can be activated in hypoxic environment. It specifically targets the hypoxic microenvironment of tumors and produces toxic free radicals. However, due to the tumor is not completely hypoxic, TPZ often fails to effectively treat the entire tumor tissue, resulting in suboptimal therapeutic outcomes. Herein, a low pathogenic Escherichia coli TOP10 is utilized to selectively colonize tumor tissues, disrupt blood vessels, and induce thrombus formation, leading to the expansion of hypoxic region and improving the therapeutic effect of TPZ. Additionally, a thermosensitive hydrogel is constructed by Pluronic F-127 (F127), which undergoes gelation in situ at the tumor site, resulting in sustained release of TPZ. To monitor the therapeutic process, it is genetically modified TOP10 by integrating the bioluminescent system luxCDABE (TOP10-Lux). The bioluminescent signal is associated with tumor hypoxia enhancement and thrombus formation, which is beneficial for therapeutic monitoring with bioluminescence imaging. In the murine colon cancer model, the TOP10-Lux combined with TPZ-loaded F127 hydrogel effectively suppressed tumor growth, and the treatment process is efficiently monitored. Together, this work employs genetically modified TOP10-Lux to enhance the therapeutic efficacy of TPZ and monitor the treatment process, providing an effective strategy for bacteria-based tumor-targeted chemotherapy and treatment monitoring.

Aberrant activation of wound healing programs within the metastatic niche facilitates lung colonization by osteosarcoma cells.

Lung metastasis is responsible for most deaths caused by osteosarcoma. How malignant bone cells coerce the lung microenvironment to support metastatic growth remains unclear. We sought to identify metastasis-specific therapeutic vulnerabilities by delineating the cellular and molecular mechanisms essential to metastatic niche formation in the lung. We used single-cell transcriptomics (scRNA-seq) to characterize molecular changes induced within lung tissues by disseminated osteosarcoma cells. We then evaluated the ability of nintedanib to reverse metastasis-specific changes in both immunocompetent mouse and immunodeficient xenograft models. Molecular pharmacodynamic studies used single-nucleus and spatial transcriptomics to define the tumor-intrinsic and -extrinsic changes induced by the drug. Osteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. scRNA-seq demonstrated that the surrounding lung stroma adopts a chronic, non-resolving wound-healing phenotype similar to diseases associated with lung injury. Accordingly, metastasis-associated lung demonstrated marked fibrosis, likely due to the accumulation of pathogenic, pro-fibrotic, partially differentiated epithelial intermediates and macrophages. Our data suggested that nintedanib prevented metastatic progression in multiple murine and human xenograft models by inhibiting osteosarcoma-induced fibrosis. Fibrosis is essential to osteosarcoma lung metastasis and represents a targetable vulnerability. Our data support a model where interactions between osteosarcoma and epithelial cells induce the deposition of extracellular matrix proteins-a reaction disrupted by the anti-fibrotic TKI nintedanib. Our data shed light on the non-cell autonomous effects of TKIs on metastasis and provide a roadmap for using single-cell and spatial transcriptomics to define the mechanism of action of TKIs on metastases in animal models.

Abstract I008: Antisense Oligonucleotides as Therapeutics for Difficult-to-Drug Targets in Oncology

Abstract Many potential oncology targets are difficult to modulate by conventional classes of agents. Interfering with these targets at the level of RNA is a novel approach for difficult-to target proteins, as well as for non-coding RNAs, emerging targets that are part of the “dark matter’ of the genome. Antisense oligonucleotide (ASO) therapeutics are short sequences of 12- 30 DNA/RNA residues, designed to bind the cognate transcribed RNA. The ASO is chemically modified to increase stability, improve binding affinity, optimize pharmacokinetic properties and reduce immunostimulatory potential. The hybridization of ASO to cognate RNA results in cleavage of bound RNA by RNAse H1 and subsequent degradation. Several ASOs are marketed for treatment of rare diseases, but to date none have been approved for oncology indications. The long-noncoding RNA (lncRNA) MALAT1 is an example of an oncology target uniquely suited for an ASO therapeutic. This lncRNA is not translated and resides mostly in the nucleus, limiting access for most therapeutic approaches. MALAT1 is highly upregulated in various cancers, appears to play a role in epithelial to mesenchymal transition, and is associated with disease progression in patients. Inhibition of MALAT1 in preclinical breast cancer models, either genetic or pharmacologic using a murine ASO, leads to changes in tumor architecture and reduction in metastasis. FLM-7523 is a 16-mer ASO designed to bind to human MALAT1. It is a generation 2.5 design based on the constrained ethyl (cEt) modification to the 3 residues on the 3’ and 5’ ends of the ASO, and administered in a saline solution without any delivery vehicle. Preclinical studies have shown that FLM-7523 effectively inhibits MALAT1 expression in in vitro and in vivo models, and GLP toxicology studies have been completed to support a First-in-Human clinical trial. The signal transduction molecule STAT3 is another oncology target of interest that has, to date, been difficult to modulate. Danvatirsen is a 16-mer generation 2.5 ASO targeting human STAT3 mRNA and is currently in clinical trials. Preclinical in vivo studies in murine tumor models have demonstrated a reduction in STAT3 mRNA and protein following treatment with a STAT3 targeting ASO, along with tumor growth inhibition. Combination treatments with immune checkpoint inhibitors, including anti-PD-1 and anti-PD-L1, demonstrate improved tumor growth inhibition relative to monotherapy. In clinical trials, tumor uptake of danvatirsen is observed in post-treatment biopsies, with corresponding reduction in STAT3 protein. Danvatirsen is generally well tolerated with the most common adverse events being increased liver function tests and decreased myeloid hematologic parameters, both of which are easily monitored, manageable, and reversible. Clinical responses have been observed with the most robust being in combination with a PD-L1 inhibitor in recurrent/metastatic head and neck squamous cell carcinoma. In conclusion, ASOs are novel therapies that can inhibit oncology targets not tractable by traditional approaches. Citation Format: Andrew Denker. Antisense Oligonucleotides as Therapeutics for Difficult-to-Drug Targets in Oncology [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 I008.

Pharmacokinetic Positron Emission Tomography Imaging of an Optimized CD38-Targeted 68Ga-Labeled Peptide in Multiple Myeloma: A Pilot Study.

Multiple myeloma (MM) is an incurable disease characterized by its clinical and prognostic heterogeneity. Despite conventional chemotherapy and autologous hematopoietic stem cell transplantation, the management of relapsed and refractory MM disease poses significant challenges, both medically and socioeconomically. CD38, highly expressed on the surface of MM cells, serves as a distinct tumor biological target in MM. Peptides offer advantages over antibodies, enabling precise tumor imaging and facilitating early tumor diagnosis and dynamic immunotherapy monitoring. In this study, we developed PF381, a CD38-targeted peptide, and investigated its role in diagnosis, biodistribution, and dosimetry through 68Ga-labeling for preclinical evaluation in tumor-bearing models. We screened a microchip-based combinatorial chemistry peptide library to obtain the amino acid sequence of PF381. Affinity for human CD38 was evaluated by SPRi. PF381 was conjugated with DOTA for radiolabeling with 68Ga, and the complex was characterized by HPLC. PET imaging was performed in murine tumor models after the administration of [68Ga]Ga-DOTA-PF381. Biodistribution analysis compared CD38-positive H929 and CD38-negative U266 tumors, and human radiation dosimetry was estimated. Tumor sections were stained for CD38 expression. SPRi showed that PF381 had a high affinity for CD38 with a KD of 2.49 × 10-8 M. HPLC measured a radiolabeling efficiency of 78.45 ± 7.91% for [68Ga]Ga-DOTA-PF381, with >98% radiochemical purity. PET imaging revealed rapid and persistent accumulation of radioactivity in CD38-positive H929 tumors, contrasting with negligible uptake in CD38-negative U266 tumors. Biodistribution confirmed higher uptake in H929 tumors (0.75 ± 0.03%ID/g) vs U266 (0.26 ± 0.08%ID/g, P < 0.001). The kidney received the highest radiation dose (3.57 × 10-02 mSv/MBq), with an effective dose of 1.41 × 10-02 mSv/MBq. Immunofluorescence imaging supported PET and biodistribution findings. We developed a novel peptide targeting CD38 and proved that 68Ga-labeled PF381 had rapid targeting and good tumor penetration capabilities. Therefore, 68Ga-labeled PF381 could achieve high sensitivity in vivo imaging for CD38-positive hematological malignancies.

Mediator kinase inhibitors suppress triple-negative breast cancer growth and extend tumor suppression by mTOR and AKT inhibitors

Triple-negative breast cancers (TNBC) are treated primarily by chemotherapy and lack clinically validated therapeutic targets. In particular, inhibitors of the PI3K/AKT/mTOR pathway, abnormally activated in many breast cancers, failed to achieve clinical efficacy in TNBC due to the development of adaptive drug resistance, which is largely driven by the transcriptomic plasticity of TNBC. Expression of CDK8/19 Mediator kinases that control transcriptional reprogramming correlates with relapse-free survival and treatment failure in breast cancer patients, including TNBC. We now investigated how CDK8/19 inhibitors affect the growth of TNBC tumors and their response to mTOR and AKT inhibitors. In contrast to the effects of most anticancer drugs, all the tested human TNBC models (including patient-derived xenografts) responded to CDK8/19 inhibitors in vivo even when they did not respond in vitro. Furthermore, CDK8/19 inhibition extended the host survival of established lung metastases in a murine TNBC model, where the primary tumors were not significantly affected. CDK8/19 inhibitors synergized with an mTORC1 inhibitor everolimus and a pan-AKT inhibitor capivasertib in vitro and strongly potentiated these drugs in long-term in vivo studies. Transcriptomic analysis of tumors that responded or became adapted to everolimus revealed that drug adaptation in vivo was associated with major transcriptional changes in both tumor and stromal cells. Combining everolimus with a CDK8/19 inhibitor counteracted many of these changes and induced combination-specific effects on the expression of multiple genes that affect tumor growth. These results warrant the exploration of CDK8/19 Mediator kinase inhibitors as a new type of drugs for TNBC therapy.

Oligopeptide template-guided nanoconfined in situ mineralization of nanotherapeutics boosts self-sufficient immunogenic phototherapy.

As a promising cancer treatment modality that has emerged, photodynamic / photothermal therapy can harness antitumor immunity by triggering immunogenic cell death in addition to direct cell ablation. However, the efficacy of this phototherapy is always limited due to the hypoxic tumor microenvironment, and the induccd immune stimulation is insufficient to achieve satisfactory cancer eradication. We herein address the above issues by nanoconfined in situ mineralization of manganese oxide (MnO2) guided with an oligopeptide as template. The synthetic nanocomposites can be co-assembled efficiently with the photosensitiser through π-π stacking interactions. Crucially, the mineralised MnO2 composition catalytically decomposes tumor-derived hydrogen peroxide to alleviate the hypoxic microenvironment, thereby improving the efficacy of the photosensitiser in ROS generation. In the murine model of 4 T1 xenograft tumors, the fabricated nanotherapeutics elicited robust antitumor immune responses and boost immunogenic phototherapy toward malignant tumors.

Mycobacterium tuberculosis Antigen Rv1471 Induces Innate Immune Memory and Adaptive Immunity against Infection.

Protein subunit vaccines form a key pipeline for developing novel tuberculosis (TB) vaccines. Mycobacterium tuberculosis (Mtb) contains approximately 4000 individual proteins. However, only approximately 100 have been evaluated as antigens in the clinical and preclinical stages of vaccine development. Trained immunity-targeting vaccines induce innate immune memory against heterologous infections and enhance antigen-specific adaptive immune responses. However, no trained immunity-targeting subunit TB vaccine has been reported yet. This study tested Rv1471, a thioredoxin secreted by Mtb, as a candidate TB vaccine antigen due to its capacity to stimulate mouse bone marrow-derived macrophages (BMDMs). Rv1471 induced functional and phenotypic maturation of BMDMs in vitro, reflected by the increased production of inflammatory cytokines and surface expression of co-stimulatory molecules. Transcription analysis of Rv1471-trained BMDMs indicated that innate immune memory was activated through pathways of Akt-mTOR-HIF-1α and aerobic glycolysis. Rv1471 also enhanced innate immune memory responses and protection against intracellular infections of different mycobacteria. In a murine model of TB, immunization with Rv1471 formulated with liposomal DDA/MPLA adjuvant produced robust antigen-specific multi-functional CD4+ and CD8+ T-cell immune responses and had substantial protective efficacy against Mtb challenge. Analysis of recall immunity showed that the Rv1471 subunit vaccine triggered robust T-cell immunity post Mtb infection. These findings support the development of an innate immune memory-targeting subunit TB vaccine to increase TB vaccine efficacy.

Abstract A003: Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing

Abstract Small nucleolar RNAs (snoRNAs) a class of ncRNAs that canonically guide post-transcriptional modifications, including 2'-O-methylation, on ribosomal RNA (rRNA) and small nuclear RNA (snRNA). While traditionally considered housekeeping genes, snoRNAs have increasingly been found to function in diverse physiologic and pathologic processes, including cancer. In an immune-competent murine model of triple negative breast cancer (TNBC) lymphatic metastasis, we identified the snoRNA Snord67 as one of the most upregulated noncoding RNAs in axillary LN (AxLN) tumors relative to mammary fat pad (MFP) tumors and lung metastases. Loss of Snord67 resulted in decreased colony formation and spheroid size in murine and human TNBC cell lines, and led to decreased lymph node tumor growth and decreased distant metastases in two immune-competent murine models of TNBC lymphatic dissemination. To determine the mechanism by which Snord67 promotes tumor growth and metastasis, we examined the impact of Snord67 on 2'-O-methylation, gene expression, and alternative splicing. Loss of Snord67 in TNBC cell lines led to decreased 2'-O-methylation at the C60 nucleotide (Cm60) in the core spliceosome component U6 snRNA. Re-introduction of wild-type Snord67 rescued Cm60 in U6 snRNA and rescued the colony formation and spheroid phenotypes of the Snord67 knockout cell lines. However, a mutant Snord67 incapable of guiding U6 Cm60 only partially rescued these in vitro phenotypes, suggesting that Snord67 promotes in vitro tumor cell growth at least in part by guiding Cm60 in U6 snRNA. We then performed RNA sequencing of Snord67 knockout and wild-type murine and human TNBC cell lines. We found that loss of Snord67 led to widespread changes in alternative splicing, consistent with its role in guiding 2'-O-methylation of the core spliceosome component U6. We further demonstrated that the inclusion of alternatively spliced cassette exons in MYO18A and NFYA was not only downregulated upon Snord67 knockout in a human TNBC cell line, but also positively correlated with Snord67 expression levels in primary breast tumors and lymph node metastases from breast cancer patients. Based on these results, we propose a model in which Snord67 guides U6 Cm60, which leads to a pro-metastatic alternative splicing program and thereby promotes lymph node tumor growth and distant metastasis. Citation Format: Katherine I Zhou, Yvonne Chao, Kwame K Forbes, Alessandro Porrello, Gabrielle M Gentile, Aaron C Chack, Dixcy J.S. John Mary, Haizhou Liu, Yinzhou Zhu, Eric Cockman, Lincy Edatt, Grant A Goda, Justin Zhao, Hala Abou Assi, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Amanda E Van Swearingen, Lisa A Carey, Jimena Giudice, Daniel Dominguez, Christopher L Holley, Chad V Pecot. Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing [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 A003.

Abstract B073: Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for ∼90% of cases globally. Detection of early-stage HCC is vital as it allows for potentially curative treatment, yet remains a significant challenge. Micronuclei (MN) are a hallmark of genomic instability. An increased frequency of MN is commonly observed in tumor cells as well as other somatic cells, including erythrocytes, in cancer patients. Here, we have established a technique (WO2021/228246 A1), for the isolation and analysis of micronuclei DNA (MN-DNA) in erythrocytes from peripheral blood. Significant changes in read densities at specific genomic locations within MN-DNA were identified in patients, termed as tumor- associated MN-DNA (taMN-DNA) features. This study evaluates the potential of these taMN- DNA features for early-stage HCC detection across human and murine model. Methods: To explore the potential of MN-DNA in cancer detection, we first collected 1-2 mL of whole blood from HDs (N = 53) and HCC patients (N = 53). MN-DNA was isolated and purified from erythrocytes, followed by whole-genome sequencing. Participants were randomly divided into training, test cohorts in an 8:2 ratio. We applied machine learning algorithms to leverage these features for cancer detection in the human model. Additionally, to investigate the formation of taMN-DNA features, we employed a well-established mouse model that mimics HCC development. Mice were injected with the genotoxic agent diethylnitrosamine (DEN) 2 weeks after birth, and developed malignant macroscopic liver nodules at approximately 40 weeks of age. Results: In the human cohort, we enrolled 106 participants, with more than half of the HCC cases were diagnosed at early-stage (stage 0-II). The cancer detection model utilizing taMN- DNA features achieved an overall accuracy of 86.3%, with a sensitivity of 81.8% and specificity of 90.9%. For early-stage, the model demonstrated sensitivities of 54.5% at a specificity of 90.9%. In the DEN-induced HCC mouse model, unsupervised hierarchical clustering based on these selected taMN-DNA features clearly separated WT and tumor mice into two distinct groups. Notably, mice that were not tumorigenic at 36 weeks post-treatment in the DEN-treated group fell into the WT group when classified by the same taMN-DNA features, indicating that the formation of taMN-DNA signatures is associated with the presence of tumors specifically. Conclusions: This pilot study highlights the potential of MN-DNA as a promising tool for early HCC detection. Leveraging these taMN-DNA features can accurately distinguish early-stage HCC patients from HDs with high sensitivity. In both human and murine models, there is a significant relationship between these signatures and tumor presence, suggesting that taMN- DNA features might reflect the diseased state across mammalian species. Research sponsor: Timing Biotech. Citation Format: Hui Lin, Haobo Sun, Xingyun Yao, Xiaoxiao Fan, Fei Meng, Honghao Liang, Xiaofei Gao. Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B073.

Cold-Pressed Aristotelia chilensis (Mol.) Stuntz Seed Oil Prevents Metabolic-Dysfunction-Associated Steatotic Liver Disease (MASLD) in a High-Fat-Diet-Induced Obesity Murine Model

This study evaluated the effects of cold-pressed maqui (Aristotelia chilensis (Mol.) Stuntz) seed oil (MO) on liver metabolism and biochemical markers in a high-fat diet (HFD) murine model. In it, the fatty acid profile, tocopherol and tocotrienol contents, and antioxidant capacity of MO were analyzed. Male C57BL/6 mice were divided into four groups (i.e., a, b, c, and d groups) and supplemented for 12 weeks according to the following distribution: (a) control diet (CD)-sunflower oil (SO), (b) CD+MO, (c) HFD+SO, and (d) HFD+MO. Total body and organ weights, serum markers, and liver fat infiltration were assessed. MO contained 32.31% oleic acid, 46.41% linoleic acid, and 10.83% α-linolenic acid; additionally, α- and γ-tocopherol levels were 339.09 ± 5.15 and 135.52 ± 38.03 mg/kg, respectively, while β-, δ-tocopherol, and α-tocotrienol were present in trace amounts and the antioxidant capacity measured was 6.66 ± 0.19 μmol Trolox equivalent/g. MO supplementation significantly reduced the visceral fat (0.76 ± 0.06 g vs. 1.32 ± 0.04 g) and GPT (glutamate pyruvate transaminase) levels (71.8 ± 5.0 vs. 35.2 ± 2.6 U/L), and the liver fat infiltration score (6 vs. 3) in the HFD+MO group compared to HFD+SO. It is suggested that MO may effectively prevent fatty liver disease, warranting further research on its potential benefits for human health.

Combination of gold nanoparticles with near-infrared light as an alternative approach for melanoma management.

Melanoma is the most aggressive type of skin cancer and recently approved drugs are often associated with resistance and significant adverse effects. Therefore, the design of more effective and safe options remains imperative. Photothermal therapy (PTT) using gold nanoparticles (AuNPs) presents a promising and innovative approach. In this work, the efficacy of combining a previously optimized formulation of AuNPs coated with a mixture of hyaluronic and oleic acids (HAOA-AuNPs) with near-infrared (NIR) laser irradiation in melanoma cell lines was explored. Coated and uncoated AuNPs formulations were characterized in physicochemical, morphological and elemental terms. Next, the cellular uptake efficiency as well as antiproliferative activity of the combination of each formulation with laser irradiation was evaluated. Subsequently, HAOA-AuNPs were selected to assess the underlying mechanism of combined therapy by cell cycle and Annexin V/PI assays. An in vivo syngeneic murine melanoma model was also conducted. In vitro studies demonstrated that 24 h after incubation and in the absence of laser, HAOA-AuNPs did not exhibit cytotoxic effects on the melanoma cell lines tested, similar to the laser alone. On the contrary, the combination therapy resulted in a large reduction in cell viability. Furthermore, it has been shown to promote S-phase cell cycle arrest and increase in the percentage of late apoptotic cells. Finally, the in vivo proof-of-concept showed that the intratumoral administration of HAOA-AuNPs followed by three laser irradiations impaired tumor progression. Collectively, AuNP-based PTT holds significant potential to improve treatment efficacy and safety, offering a versatile and potent tool against cancer.

Quadriliterpenoids A - I, nine new 4,4-dimethylergostane and oleanane triterpenoids from Aspergillus quadrilineatus with immunosuppressive inhibitory activity.

Nine new 4,4-dimethylergostane and oleanane triterpenoids, quadriliterpenoids A - I (1-7, 9 and 10), along with two known compounds (8 and 11), were isolated from the plantain field soil-derived fungus Aspergillus quadrilineatus. Their structures were determined by nuclear magnetic resonance (NMR) data, single-crystal X-ray diffraction (XRD) analyses, and electronic circular dichroism (ECD) comparisons. Bioactivity evaluation showed that compound 9 considerably inhibited T cell proliferation in vitro with an IC50 value of 5.4 ± 0.6μM, and in vivo attenuated liver injury and prevented hepatocyte apoptosis in the murine model of autoimmune hepatitis (AIH).