Oncotarget

Emerin (EMD), an inner nuclear membrane protein essential for nuclear architecture integrity, gene expression, cellular signaling, and chromatin stability, interacts with the LINC complex and participates in cytoskeleton-nucleoskeleton communication by binding to nuclear actin filaments. EMD is implicated in migration, invasion, and metastasis in some tumors, but its role in glioblastoma (GBM) remains unclear. This study evaluated the effects of EMD knockdown and overexpression in GBM cell lines following genotoxic treatment with cisplatin. In both wild-type p53 (U87-MG) and mutant p53 (U138-MG) GBM cells, EMD expression is high, and cisplatin treatment did not affect these protein levels. EMD knockdown in U87-MG cells significantly increased cisplatin IC50, viability, and proliferation. Conversely, stable overexpression of EMD in U87-MG cells led to reduced cisplatin IC50, viability, proliferation, and migration. EMD knockdown or overexpression did not affect any U138-MG phenotypes, with or without cisplatin treatment. Modulation of EMD levels causes morphological changes in stress fiber cytoskeleton, whereas overexpression of EMD in U87-MG cells promotes an increase and a decrease in nuclear and cytoplasmic actin levels, respectively. These biological responses of U87-MG cells overexpressing EMD were coincidentally associated with alterations in the levels of pH2AX(Ser139), p-p53(Ser15), p53, and p21Kip1 proteins after cisplatin exposure. In sum, modulation of EMD levels affects the viability, migration, and proliferation of wild-type p53 GBM cells treated with cisplatin, suggesting unknown roles in the DNA damage response and repair. This work highlights EMD as a potential regulator of GBM chemoresistance and a target for therapeutic intervention.

Canine hemangiosarcoma (HSA) is an aggressive endothelial malignancy with limited therapeutic options, and its progression is closely associated with vascular architecture, stromal remodeling, and inflammatory cell recruitment. Sulfoquinovosylacylpropanediol (SQAP) is a sulfoquinovosyl lipid radiosensitizer reported to affect angiogenic and tumor-microenvironmental pathways, but its effects in canine HSA are unknown. Here, we evaluated SQAP in canine HSA cell lines and patient-derived xenograft (PDX) models. SQAP showed minimal direct cytotoxicity against HSA cell lines in vitro, whereas it significantly suppressed tumor growth in three canine HSA PDX models. Transcriptome analysis of SQAP-treated HSA PDX tumors detected more SQAP-responsive genes in mouse host-derived cells than in canine tumor cells. Gene-set enrichment analysis of the mouse host-derived fraction showed positive enrichment of angiogenesis, hypoxia, and stromal remodeling-related gene sets after SQAP treatment. Subsequent tissue analysis showed that SQAP reduced host-derived CD31-positive vascular area and increased -smooth muscle actin coverage of remaining vessels in two of the three PDX models, while altering macrophage-associated marker profiles in a model-dependent manner. These findings indicate that SQAP suppresses canine HSA PDX growth primarily through vascular and macrophage-associated remodeling of the tumor microenvironment rather than direct tumor-cell cytotoxicity.

Background: Medulloblastoma (MB) is the most common malignant brain tumour in children, and aggressive subgroups are frequently driven by the oncoproteins MYC or MYCN. Direct therapeutic targeting of MYC/MYCN has been challenging because of their intrinsically disordered protein structures. The aim of this study was to determine whether novel SE486-11 analogues (UNSW-SCs) can therapeutically target MYC/MYCN-driven MB. Methods: The anticancer activity of UNSW-SCs was assessed in MB cell lines with differential MYC/MYCN expression. Target engagement was evaluated using surface plasmon resonance and drug affinity responsive target stability assays. Blood-brain barrier penetration, MYC/MYCN protein degradation, cell cycle effects, apoptosis, DNA damage, and synergy with histone deacetylase (HDAC) inhibitors were examined. Therapeutic efficacy was evaluated in murine models of MYC- and MYCN-driven human MB. Results: UNSW-SCs showed potent anticancer activity, with preferential selectivity toward MB cells expressing high MYC/MYCN levels and IC50 values ranging from 0.22 to 1.18 M. The lead molecule, UNSW-SC-22, directly bound MYC, crossed the blood-brain barrier, and achieved a brain-to-plasma ratio of 1.44 at peak concentrations. UNSW-SC-22 induced MYC/MYCN-dependent cytotoxicity associated with enhanced proteasomal degradation, cell cycle arrest, apoptosis, and DNA damage. Combined treatment with HDAC inhibitors further reduced MYC/MYCN protein levels, increased DNA damage, and enhanced apoptosis. In vivo, UNSW-SC-22, either alone or with entinostat, significantly suppressed intracranial tumour growth and prolonged survival. Conclusions: UNSW-SC-22 is a brain-penetrant MYC/MYCN-targeting molecule with potent preclinical activity in MYC/MYCN-driven MB, supporting its development as a monotherapy or combination strategy with HDAC inhibition.

BackgroundReactive Oxygen Species (ROS) are the small, unstable and highly reactive species, having DNA oxidizing ability. Oxidation of the DNAs purine and pyrimidine bases can lead to single or double strands in this macromolecule. In this situation, the ATM molecule, a serine-threonine kinase, targets several proteins for phosphorylation, which causes the cell cycle to stop and the DNA damage repair begins. It has previously been proven that natural polyphenols have the cancer inhibiting properties due to their high efficacy and low side effects. Silibinin is the main herbal and medical ingredient in Milk Thistle (Silybum marianum) is a polyphenol flavonolignan, which has been widely considered as an antioxidant and anticancer agent. The purpose of the present study was to investigate the ATM gene expression and measurement of reactive oxygen species (ROS) in SKBR3 cell line, treated with Silibinin. Materials and MethodsAt first, the SKBR3 cell line was cultured in RPMI1640 culture medium and MTT assay was carried out to evaluate the Silibinin cytotoxicity. Flow Cytometry was carried out for cell cycle analysis, apoptotic induction, and ROS detection. While, Real Time PCR was used to evaluate the ATM gene expression in the Silibinin-treated and un-treated SKBR3 cells. ResultsPresent results have shown that 150 {micro}M Silibinin had the most significant cytotoxicity and apoptotic induction influence after the treatment period of 48 h. Flow cytometry data have shown that Silibinin induced considerable amount of apoptosis and caused cell cycle arrest at G1/S phase and induced production of ROS. Real-time PCR results have revealed that Silibinin increased the ATM expression in SKBR3 cell line. ConclusionSilibinin causes increased ATM gene expression by inducing ROS production, which initiates cell cycle arrest and apoptotic induction in SKBR3 cells line.

Malignant brain tumors are among the most aggressive and difficult to treat human cancers. Glioblastomas (World Health Organization grade IV gliomas) are particularly lethal and refractory to treatment. Few drugs exist that are even somewhat effective. Our investigation of the physiologic role of fatty acid (FA) activating enzymes (acyl-CoA synthetase; ACS) identified an ACS that was widely expressed in gliomas but not in normal glial cells. Depletion of this enzyme, ACSVL3 (very long-chain ACS3), by knockdown or knockout decreased the malignant behavior of several glioma cell models including U87MG and Mayo-22 cells both in culture and when grown as xenografts. Hypothesizing that ACSVL3 is a potential therapeutic target in glioma, we conducted a search for inhibitors of this enzyme and found that CB5 (grassofermata) was a promising candidate. Treating U87MG glioma cells with CB5 slowed growth in monolayer culture; the growth rate was similar to that seen in cells in which ACSVL3 was either knocked down or knocked out. CB5 inhibited growth in a dose-dependent manner over a narrow range, and concentrations above 10 M were toxic. Treatment at the lower dose of 3 M inhibited growth of U87MG cells but was reversible, suggesting that this dose was not toxic. CB5- treated U87MG cells exhibited an altered morphology with a larger size and longer projections. In contrast, normal human fibroblasts treated with 10 M CB5, a concentration that was toxic to U87MG cells, showed no effect on either growth rate or morphology. Treating U87MG cells with 3 M CB5 induced differentiation as shown by increased expression of the astrocyte-specific marker glial fibrillary acidic protein (GFAP). In contrast, GFAP levels remained low in ACSVL3 knockdown cells. CB5- treated U87MG cells were less invasive, and thus less malignant, than either untreated cells or ACSVL3 knockout cells when assessed by a scratch wound healing assay. Acute treatment of U87MG cells with 3 M CB5 decreased the ability of these cells to degrade FA of differing chain lengths from 16-24 carbons by {beta}-oxidation, suggesting that decreased ACS enzyme activity contributes at least in part to the drugs mechanism of action. NOD/SCID mice receiving up to 32 mg/kg/day CB5 by intraperitoneal injection showed no obvious side effects, suggesting that the drug was well-tolerated. Xenografts induced by subcutaneous injection of U87MG cells in the flanks of NOD/SCID mice were allowed to grow for 8 days after which half of the mice were treated with 2 mg/kg/day CB5. After 7 days of treatment, xenograft growth slowed in the treated mice and by 12 days tumor size had begun to decrease, suggesting therapeutic efficacy. When a similar study was done using xenografts induced by subcutaneous injection of Mayo-22 cells, which are maintained as subcutaneous tumors in mice rather than in cell culture, the effect of CB5 on tumor growth or weight at sacrifice was not statistically significant. The results of these studies suggest that CB5 may have therapeutic value in malignant glioma. Additional studies using other glioma models and other drugs chemically related to CB5 seem warranted.

Analyses of human breast carcinomas (BCs) and premalignant breast lesions show that the loss of RASSF1A is an early event in the development of ER+ BCs, which correlates linearly with malignant progression. This observation suggests that RASSF1A inhibition is important for the development and progression of ER+ BCs. In addition to RASSF1A, concurrent caveolin-1 (Cav-1) inhibition may further promote ER+ breast carcinogenesis. In the present study, transgenic Rassf1a-/- and Cav-1(-/-) single as well as Rassf1a-/-, Cav-1(-/-) double knockout mice were used to investigate the impact of single or combined Rassf1a and Cav-1 inactivation on BC initiation. Loss of either one or both proteins led to different, pre-malignant histopathological alterations within the mammary glands of the mice, but not to fully developed BC, confirming that Rassf1a and Cav-1 are both important for maintaining the integrity of mammary gland epithelial structure, but suggesting that further intracellular changes or extracellular factors are required for the development of luminal BC when both genes are lost.

Background. Sentinel lymph node biopsy (SLNB) is an essential component of axillary staging in breast cancer. Fluorescence-guided mapping with indocyanine green (ICG) enables real-time visualization of lymphatic drainage; however, the parameters of fluorescence-signal recording require standardization. Objective. To assess the technical feasibility and clinical applicability of SLNB with ICG under near-infrared (NIR) imaging guidance in patients with breast cancer. Materials and Methods. This prospective single-center study included 30 patients who underwent ICG-guided SLNB between 2023 and 2025. In 24 patients, ICG mapping was combined with technetium-99m radioisotope navigation; in 6 patients, ICG guidance alone was used. The protocol comprised periareolar ICG injection, standardized imaging conditions, and fluorescence-index recording. Results. The protocol was completed in all 30 patients. Fluorescent visualization of the lymphatic pathway and/or the sentinel lymph node (SLN) was achieved in every case, and no ICG-related adverse reactions were recorded. The mean fluorescence index was 213.0 +/- 24.7, the median was 206.0 [192.5-237.2], and the min-max was 180-255. Conclusion. SLNB with ICG under NIR imaging guidance demonstrated technical feasibility in a prospective single-center cohort. Quantitative fluorescence-index recording may serve as a component of standardizing intraoperative fluorescence guidance. Keywords: breast cancer; sentinel lymph node biopsy; indocyanine green; near-infrared imaging; fluorescence lymphography; fluorescence index; axillary staging.

Abstract Background: Acute Lymphoblastic Leukemia (ALL) is a highly heterogeneous pediatric malignancy. Despite high survival rates, relapse and the involvement of central nervous system (CNS) remains a significant clinical challenge. Traditional clinical parameters often lack the precision required for early detection and risk stratification. This study utilizes high-throughput proteomics and machine learning to identify molecular signatures in cerebrospinal fluid (CSF) that characterize disease effect and treatment response. Methods: 82 CSF samples from 41 pediatric ALL patients at diagnosis (VD) and remission (VR) were analyzed. Proteomic profiling of 276 proteins was performed using Olink Proximity Extension Assay. Differentially abundant proteins were identified (q-value< 0.05, |Log_2FC| > 0.5) using the Wilcoxon rank-sum test. Three machine-learning algorithms - Random Forest, LASSO, and SVM-RFE - were integrated to select the differentially abundant proteins in VR and VD and between CNS involvement levels. To validate the data Pan-Cancer Atlas analysis was done using two different platforms. Results: In the remission phase, we observed significant alterations in the expression of key proteins compared to diagnosis, with ADGRG1 and KYNU showing a marked increase, while CCL17, CD5, CD27, CXCL9, CXCL11, FASLG, GZMA, and TNFRSF9 were significantly downregulated. Furthermore, our analysis identified distinct protein signatures associated with CNS involvement: CCL4, CTSC, CXCL10, CXCL9, and MMP7 were differentially abundant at the VD stage, whereas CAIX, CASP-8, HAGH, CXCL9, MMP7, MCP-2, and VWC2 at the VR stage. Conclusion: Integrating Olink proteomics with machine learning identified molecular signatures in ALL that have the potential to be further developed to a biomarker panel for monitoring treatment response and guiding personalized therapeutic strategies shifting the focus toward the Precision One Health approaches.

Thymosin {beta}4 (T{beta}4) is a conserved acidic polypeptide with 43-amino acids participating in multiple pathophysiological processes. In this study in vivo effects of T{beta}4 on liver regeneration are investigated in carbon-tetrachloride (CCL4) induced rodent animal liver jury models. Results illustrate that exogenous T{beta}4 treatment significantly reduced CCL4-rendered liver necrosis around central vein. At 48 hours after CCL4 insults hepatocytes proliferation occur mainly around the periportal area, while hepatocytes proliferation around the necrosis area is prominently increased by exogenous T{beta}4 treatment. The holistic proliferation level of liver tissues are also enhanced by exogenous T{beta}4. Hepatocyte proliferation activities negatively correlate with the necrosis extent of the liver tissue. These results suggested firstly exogenous T{beta}4 treatment could enhance liver regeneration and exhibit prosperous potential for application in clinical conditions such as liver transplantation.

Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy posing significant diagnostic challenges, particularly in distinguishing it from other adrenal tumors, such as adenoma and pheochromocytoma, due to overlapping imaging and biochemical features. Improved non-invasive tools are critically needed for earlier, more accurate classification of this rare cancer. This pilot study analyzed serum lipidomic profiles in ACC, pheochromocytoma, and adenoma patients versus healthy volunteers. The most significant alterations occurred in sphingomyelins (SM) and diacylglycerols (DG). All tumor samples showed reduced very-long odd-chain SM (e.g., SM 39:1, SM 41:1, SM 41:2) and elevated DG (e.g., DG 34:1, DG 34:2, DG 36:2). These abnormalities were most pronounced in malignant tumors: ACC and metastases (AUC = 0.933), followed by pheochromocytoma (AUC = 0.800) and adenoma (AUC = 0.711). ACC patients also exhibited specific lipid signatures with decreased alkyl/alkenyl phospholipids (e.g., PE O-38:5) and lysophosphatidylcholines (e.g., LPC 20:5, LPC 18:2) versus healthy volunteers, not observed in pheochromocytoma or adenomas. Ceramide species (e.g., Cer 42:2;O2, Cer 34:1;O2) were increased in ACC compared to the other tumor types. Incorporating lipid-to-lipid ratios (Cer/SM, Cer/DG) further improved statistical model accuracy. Compared to clinical biochemistry/oxidative stress (OS) parameters, lipidomic profiling showed superior discriminatory power in adrenal tumor diagnosis. The presented study shows the serum lipidomic profiling as a promising non-invasive method for distinguishing adrenal tumor subtypes (ACC, pheochromocytoma, and adenoma) from healthy individuals, with strong diagnostic potential for ACC.

Polo-like kinase 1 (PLK1), a key regulatory protein controlling entry into and passage through mitosis, has been targeted through its kinase domain (KD) with mixed clinical success. Inhibition through the Polo-box domain (PBD) is a viable alternative through targeting the sub-cellular localization and kinase activity of PLK1. Novel non-peptidic compounds, termed abbapolins, were discovered through the REPLACE strategy and have been lead optimized through structure-based strategies and screening analogs in the NCI-60 tumor cell panel. Proteomic analysis revealed a correlation between abbapolin activity and PLK1 protein levels in the cell lines part of the NCI-60. Prostate cell lines were identified as among the most sensitive and led to further detailed studies of their activity in prostate cancer models. Compounds were evaluated for their pharmacokinetic properties, and in vivo efficacy, and results showed significant antitumor xenograft activity with no observable gross toxicity. Treated tumors were analyzed for loss of PLK1, which was previously shown to be induced by abbapolin binding. Results obtained showed a significant degradation of PLK1 in abbapolin-treated vs untreated tumors, thereby confirming on-target action in vivo and revealing PLK1 levels as a potential pharmacodynamic marker. Lead compounds were shown to sensitize PC tumors resistant to androgen deprivation therapy paving the way for future combination studies in vivo. These data provide an alternative pathway for effective PLK1 therapeutics that avoid the reported problems of molecules targeting the KD, in vivo proof-of-concept for the REPLACE strategy and validation for targeting the PBD as an anti-tumor drug development strategy.

Genetic uniqueness of the tumor microenvironment significantly influences cancer growth, survival, and response to therapy, independent of the cancer cells intrinsic properties or the adaptive immune system. Using genetically distinct Rag1-/- mouse models, this study shows that different strains exhibit varied tumor growth kinetics and survival outcomes when xenografted with identical leukemic and solid tumor cell lines. This study further highlights the critical role of the myeloid immune compartment and shows that disrupting both lymphoid and myeloid systems alters cancer progression. These results also reveal that the tumor microenvironment can permanently alter cancer cell phenotypes and significantly affect chemotherapy efficacy, as seen with Cisplatins varying effects across strains. These findings underscore the importance of considering genetic background in preclinical cancer models, suggesting that reliance upon a single mouse strain may lead to incomplete conclusions about cancer biology and treatment efficacy. SUMMARY STATEMENTPre-clinical xenograft mammalian models are used to study human diseases. Here we report that the genetic uniqueness of the tumor microenvironment, independent of the immune system, can determine the fate of cancer progression, survival, and therapy response.

Background: The Prosigna Breast Risk of Recurrence test is based on the PAM50 classifier and was originally validated as an in vitro diagnostic (IVD) test on the Dx enabled nCounter(R) Analysis System. The Prosigna test is intended for early-stage, hormone receptor+ (HR+) breast cancer and provides the risk of recurrence (ROR) score (0-100), intrinsic subtype (Luminal A, Luminal B, HER2-enriched, and Basal-like), and the 10-year probability of distant recurrence. We describe the performance of the Prosigna test as a whole transcriptome RNA sequencing laboratory developed test (LDT) for measuring the Prosigna ROR score and intrinsic subtypes on tissue from surgical resection and core needle biopsy as compared to the Prosigna test on the nCounter system. Methods: We evaluated three separate breast cancer cohorts to 1) bridge the IVD test on the nCounter system and NGS LDT test (n = 245), 2) validate the bridged algorithm on an independent biobank sample set (n = 187), and 3) retrospectively test performance on long-term archival samples from a previous study (n = 109). Results: Bridging analysis showed minimal score variability and robust correlation of Prosigna ROR scoring in surgical resections (SR) (2.459, SD; 0.981, R2) and core needle biopsy (CNB) (2.338, SD; 0.970, R2) samples. In the validation set, the Prosigna NGS LDT ROR scores maintained high correlation to the scores of the nCounter system (SR = 0.968, CNB = 0.966, R2), exhibited minimal score variability (SR = 2.488, CNB = 2.558, SD), and demonstrated high concordance in subtype classifications (SR = 92.3% CNB = 92.8%). Further testing demonstrated comparable performance across tumor fractions, a lower limit of detection (LLOD) of 5 ng, and robustness to exogenous ethanol or genomic DNA contamination. When testing previously extracted RNA from the clinical cohort, we observed high correlation (0.974, R2) and low variance (3.078, SD) of ROR scores with original values on the nCounter system, along with strong risk group (95.4%) and subtype (94.5%) concordance. Conclusions: This study describes the analytical validation of the Prosigna NGS-based LDT measuring the Prosigna ROR score and intrinsic subtypes with robust analytical performance on SR and CNB specimens, providing confidence for clinicians utilizing the NGS-based version of this well-established test.

Background/ObjectivesGlioblastoma is an aggressive primary brain tumor that develops within a chronically low-oxygen microenvironment, yet most preclinical studies are performed under atmospheric oxygen conditions that poorly reflect in vivo physiology. This study investigated how sustained culture under physiological oxygen tension (physioxia; 5% O{square}) influences glioblastoma cell behavior, signaling, and therapeutic response. MethodsMultiple patient-derived glioblastoma models were cultured under normoxia (21% O{square}) or sustained physioxia (5% O{square}) for at least seven days before experimentation. Cell migration, proliferation, cell cycle distribution, expression of the epithelial-to-mesenchymal transition-associated transcription factor Slug (SNAI2), PDGFR{beta}-associated signaling, and sensitivity to 5-fluorouracil were evaluated using transwell migration assays, cell counting, flow cytometry, RT-qPCR, immunoblotting, and BrdU incorporation assays. Additional patient-derived cultures established and maintained continuously under physioxia were used to examine the effects of oxygen history. ResultsSustained physioxia consistently increased migration across all glioblastoma models while reducing proliferation in normoxia-adapted cell lines through increased G0/G1 cell cycle arrest. Physioxia significantly increased Slug expression in all models and enhanced PDGFR{beta}, AKT, and ERK phosphorylation in a cell line-dependent manner. Therapeutic sensitivity to 5-fluorouracil was also altered, with physioxia conferring increased resistance in selected glioblastoma models but not universally. Patient-derived cultures maintained continuously under physioxia retained enhanced migratory capacity and exhibited increased proliferation compared with normoxia, indicating that prior oxygen exposure influences proliferative responses while the pro-migratory phenotype remains conserved. ConclusionsPhysiological oxygen tension is a major regulator of glioblastoma cell behavior, influencing migration, proliferation, signaling, and therapeutic response. These findings demonstrate that conventional normoxic culture conditions can obscure biologically relevant phenotypes and support incorporating physioxia into experimental design to improve the physiological and translational relevance of preclinical glioblastoma research.

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer that frequently carries integrated Merkel cell polyomavirus DNA and expresses oncogenic viral small T antigen (sTAg) and truncated large T antigen (tLTAg). We previously reported a mouse model of MCC with skin-targeted expression of sTAg, tLTAg, and the Merkel cell transcription factor ATOH1, combined with deletion of Trp53. Here, we optimized this model to achieve 100% tumor penetrance with lymph node metastases, established four mouse MCC cell lines, and selected one line, mMCC2, for pilot preclinical trials. In immunocompetent C57BL/6J mice, mMCC2 cells reliably produce MCCs and lymph node metastases following subcutaneous or intradermal (orthotopic) injection, and liver and lung metastases after tail vein injection. Mouse MCC allografts resemble parental tumors histologically and express a full complement of MCC differentiation markers. Treatment of allografted mice with anti-PD-1 resulted in variable inhibition of tumor growth. In contrast, treatment with lysine-specific histone Wdemethylase 1 (LSD1) inhibitors, with or without anti-PD-1, led to consistently lower tumor volumes by 5.7-fold in both groups (P < 0.0001) and smaller or undetectable lymph node metastases. Growth-inhibited tumors in all groups showed a marked reduction in proliferating tumor cells and increased infiltration by F4/80+ macrophages and CD8+ T cells. These findings support a role for immune-cell recruitment in treatment response and underscore the importance of immunocompetent preclinical models, even in studies using targeted therapies. This unique virus-positive MCC allograft model, which produces local tumors as well as regional and distant metastases in immunocompetent hosts, provides a critical platform for preclinical evaluation of new therapeutic strategies and sets the stage for much-needed translational studies to inform future clinical trials.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

Longitudinal monitoring of circulating tumor DNA (ctDNA) has emerged as a promising framework for characterizing treatment response dynamics in cancer. Scalable tumor-naive approaches for quantifying ctDNA often involve whole-genome sequencing (WGS) or DNA methylation profiling, but their comparative performance and capacity for complementary integration remain poorly understood. Here we systematically benchmarked tumor-naive WGS- and methylation-based ctDNA quantification methods using plasma from 150 patients with colorectal, lung and breast cancer. Using paired high-depth WGS and EM-seq data, we generated 40,000 in silico samples and evaluated detection accuracy, limits of detection (LoD) and quantification (LoQ) across cancer types and sequencing depths (0.1x-30x). We further assessed single- and multimodal method combinations, identifying conditions under which integrated approaches enhance analytical performance for detection and quantification relative to single modalities. This benchmark delineates key performance trade-offs and provides a practical framework to support method development and guide future research applications in ctDNA-based biomarker studies.

Glyceraldehyde (GA) contributes to the development of various diseases, such as diabetes and Alzheimer's disease via protein glycation and the formation of advanced glycation end products (AGEs); however, effective strategies for neutralizing GA are limited. Carnosine (Car), an imidazole dipeptide (IDP) that is abundant in meat, suppresses protein glycation by scavenging reactive aldehydes. There are only a few reports on the antiglycation activity of Car against GA. For other IDPs, such as anserine, balenine (Bal), and homocarnosine, there are almost no reports on their antiglycation activity. In this study, we demonstrated the antiglycation activity of four types of IDPs and 2-oxocarnosine (2-oxo-Car), an oxidized form of Car, against GA-induced intracellular protein glycation and neuronal cytotoxicity. Car and Bal exhibited significantly higher reactivity with GA compared with other IDPs and 2-oxo-Car. An in silico analysis suggested that the difference in reactivity is dependent upon intramolecular hydrogen bond formation and the conformation of each IDP. Although there were differences in reactivity with GA, LC-MS analysis revealed that all of the IDPs and 2-oxo-Car reacted with two molecules of GA to form adducts containing pyridinium rings. Car and Bal exhibited high reactivity with GA and markedly suppressed GA-induced cytotoxicity in SH-SY5Y cells. Western blot and qPCR analyses revealed that IDPs suppressed GA-induced protein glycation and the upregulation of endoplasmic reticulum and oxidative stress response genes. Our results indicate that IDPs represent a novel preventive approach to AGE-related diseases and provide a foundation for the development of strategies to treat GA-related neurotoxicity.

Abstract (250 words) Background: Glutamine addiction is a hallmark of aggressive tumors, yet glutaminase (GLS1) inhibitor CB-839 showed disappointing anti-tumor efficacy in clinical trials. L-5-[11C]-glutamine ([11C]glutamine) PET enables non-invasive assessment of glutamine metabolism in vivo, providing a tool to test mechanistic hypotheses, and identify tumors likely to respond to GLS1 inhibition: focusing on compartmentation of GLS1-derived glutamate, CB-839 impact on flux, and reciprocal glutamine synthesis. Methods: Glutaminolytic TNBC (HCC1806) and poorly glutaminolytic ER+ (MCF-7) xenograft mice with or without CB-839, underwent dynamic [11C]glutamine PET. HPLC quantified fractional radioactivity of [11C]glutamine, soluble metabolites ([11C]glutamate, [11C]CO2), and macromolecule-incorporated metabolites from blood and tumor. A four-tissue compartment model characterized GLS1 activity (k_GLS) and flux, glutamine synthetase activity (k_GS), and subcellular glutamate distribution by comparing single vs. dual glutamate pool models. Averaged tumor curves and HPLC-derived tumor metabolites were fit. Monte Carlo simulations assessed parameter estimation performance. Results: The single glutamate pool model showed high correlations between k_GLS and other parameters, yielding inflated k_GLS estimates. The dual glutamate pool model reduced correlations, improved k_GLS recovery, and yielded subcellular glutamate distributions consistent with in vitro measurements. In TNBC, k_GLS was 3-fold higher than ER+ tumors (non-overlapping 95% CI) with glutamate concentrated in the mitochondrial compartment. CB-839 reduced k_GLS in TNBC and depleted mitochondrial glutamate (non-overlapping 95% CI), though glutaminolytic flux showed no distinguishable change. ER+ tumors showed higher k_GS compared to TNBC. Conclusion: [11C]glutamine PET kinetic analysis reveals distinct glutamine metabolic phenotypes in breast cancer subtypes. Preserved glutaminolytic flux and cytosolic glutamate in TNBC provide mechanistic hypotheses for clinical failure of GLS1 inhibitors, informing ongoing studies.

The platelet P2Y1 receptor (P2Y1R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y1R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y1R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y1R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y1R towards G12/13-mediated pathways, but not those associated by Gq activation in P2Y1R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y1R-mediated functions. In vivo, KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the neutral P2Y1R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y1Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis. Key PointsO_LIBiased inverse platelet P2Y1R agonists selectively supress inflammation whilst preserving hemostasis and the ability of platelets to aggregate. C_LIO_LIBiased inverse agonism selectively inhibited P2Y1R G12/13 (Rho-GTPAse functions) but not Gq activities (PLC functions). C_LI