Oncotarget

Rationale: Neuroblastoma is a devastating pediatric malignancy, for which surgical resection is a key factor in long-term survival. However, there are significant challenges in its resection, particularly in high-risk disease, as neuroblastoma encases surrounding critical structures, is often difficult to distinguish from desmoplastic or scar tissue, and can carry occult deposits of disease not readily identified on preoperative imaging or intraoperative visualization. Building on the principles of fluorescent and radio-guided surgery, in combination with the known overexpression of GD2 in neuroblastoma, we sought to develop and optimize 111In-Dinutuximab-IRDye800, a dual-modality GD2-targeted intraoperative molecular imaging agent, for use in pediatric neuroblastoma to help enhance patient safety while facilitating a more complete resection. Methods: Dinutuximab was conjugated to IRDye800 and DTPA, then radiolabeled with Indium-111 to yield 111In-Dinutuximab-IRDye800. Optimization occurred through ELISA assay to assess binding affinity, fluorescence intensity analysis to determine the optimal fluorescent degree of labeling, and phototoxicity testing through flow cytometry. Rodent models of neuroblastoma were then generated through injection of SK-N-BE(2) human neuroblastoma cells into the left adrenal glands of nude mice or RNU rats. A series of fluorescent and gamma biodistributions was performed, varying the dose, timing, and specific activity of the tracer. Tumor and organ uptake of the tracer was compared with one- or two-way ANOVA as appropriate, with Sidaks multiple comparison test to compare tumor uptake to individual organs. Once optimization was complete, a clinically significant events study modeled after human clinical trials was performed to evaluate the in vivo capabilities of 111In-Dinutuximab-IRDye800. Results: Increased ratios of IRDye800 per antibody led to decreased binding affinity for GD2 and was associated with formulation instability without significant return on fluorescence intensity. Specific activity of the tracer was not found to impact overall biodistribution of the tracer. A 45-50 microgram dose of 111In-Dinutuximab-IRDye800 with ratios around 1 DTPA and 1-1.5 IRDye800 per antibody imaged 4 days after tracer administration was found to be the optimal combination that maximized detectable tumor-specific signal. In the clinically significant events study mirroring human IMI clinical trials, fluorescent guidance identified additional malignant lesions not originally detected under white light in 64% of rodents. Conclusions: 111In-Dinutuximab-IRDye800 is a dual-modality GD2-targeted intraoperative imaging agent that is well-poised for clinical translation. As it preserves tumor specificity, yields clinically meaningful radiofluorescent signal, and is well-tolerated without adverse events after optimization was completed, it carries the potential to positively impact the safety and completeness of neuroblastoma resection.

BCL2L13 is a mitochondrial BCL2 family protein linked to mitophagy and ceramide metabolism, but its role in NSCLC metastatic plasticity remains unclear. Human lung cancer Tissue Microarray and matched patient specimens showed subtype and site dependent BCL2L13 expression, with higher cytoplasmic granular staining in primary NSCLC and reduced, heterogeneous staining in lymph node metastases, most evident in adenocarcinoma and squamous cell carcinoma. Because Epithelial mesenchymal transition and anoikis resistance are central requirements for metastatic dissemination, this primary to node attenuation provided the rationale to test BCL2L13 knockdown and overexpression in metastasis relevant NSCLC models. In A549 and LLC cell lines. TGF beta 1 induced coordinated mitophagy and EMT with mitochondrial enrichment of BCL2L13. BCL2L13 knockdown impaired TGF beta 1 and carbonyl cyanide m chlorophenyl hydrazone associated mitophagy, reducing LC3 beta mitochondria colocalization, TOMM20, LAMP1 overlap and mitochondrial LC3 II, p62, TOMM20 turnover; BNIP3 and NIX redistribution did not compensate. BCL2L13 loss enhanced EMT marker switching and migration, whereas overexpression partially opposed these changes. During detachment, BCL2L13 knockdown reduced anoikis associated apoptosis despite preserved mitochondrial recruitment of BAX, BAK, BNIP3,NIX, altered BID processing, non parallel caspase activity and shifted FAK phosphorylation. Pharmacological autophagy modulation did not reverse this anoikis phenotype. Lipidomics identified adhesion state dependent ceramide synthases CerS2, CerS6 linked sphingolipid remodeling: BCL2L13 knockdown increased C24 linked sphingolipid species in attached cells but reduced C16, C24 ceramide related profiles during anoikis. These findings identify BCL2L13 downregulation as a metastasis associated mitochondrial-lipid state that limits mitophagic quality control while favoring EMT and detachment survival in NSCLC adenocarcinoma.

Purpose/Objective There is paucity of data reporting outcomes of breast cancers with initial internal mammary nodal involvement and no visceral metastases, treated with curative hypofractionated radiotherapy . We report the outcomes from a tertiary centre alongside spatial patterns of recurrences in the above group Material/Methods For this retrospective cross-sectional study, consecutive patients contoured as per the ESTRO 2013 guidelines, treated between 2016-2022 were eligible if their diagnostic imaging demonstrated involvement of the internal mammary nodes. Radiotherapy (40 Gy/15#/3 weeks) was delivered to the residual breast / thoracic wall, SCF region corresponding to the ESTRO lymph node level 4 and internal mammary chain nodes. Residual IMN/ level 4 nodes received a boost of 10Gy/5#. Spatial mapping of sites of recurrence at the local site and three nodal sites (axilla, SCF and IMN) was performed using deformable image registration. Sites of recurrence at the local site and three nodal levels were contoured separately. Volumetric intersection of the recurrent gross tumour volume (GTV_recurrence) with treated clinical target volume (CTV) was calculated. Actuarial overall (OS), disease free survival (DFS) & cumulative incidence of local (LR), regional (RR) and loco-regional recurrence(LRR) were calculated using Kaplan Meier method. Univariate comparison of outcomes with or without residual disease was performed using the log rank test. Results The median age of the 61 eligible women was 49 years. 77% received neoadjuvant chemotherapy and the rest adjuvant chemotherapy. 82% patients had a mastectomy. Axillary lymph node dissection was done in 96.7%. Boosts to residual IMN and SCF nodes were delivered to 21(34.4%) and 2 (3.3%) respectively. Median follow up was 3.6 years. Out of the 61 patients, 42 patients were disease free with an estimated 3 year disease free survival of 75% (95% CI 64, 88%). Spatial mapping of locoregional recurrence was possible in all but 1 patient with local (only) recurrence who was lost to follow-up after mammogram only. Among the patients with loco regional recurrence 1 had recurrence in local site + SCF +axilla, 3 had recurrence in the SCF+axilla, 2 in the SCF+IMN and 1 in the axilla+SCF+IMN. Only one patient had isolated axillary recurrence or isolated SCF recurrence. There were no IMN only recurrences. Among the 8 patients with nodal recurrence, a total of 27 individual GTV_recurrence were identified in the axilla(n=11), SCF(n=11) and IMN (n=5). IMN recurrences showed complete or partial overlap with CTV. SCF recurrences were a mix with predominantly in-field recurrences while axillary recurrences occurred outside the treated volume.Four (6.6%) patients had Grade 2 lymphoedema as documented late side effect. Conclusion Aggressive treatment of IMN disease with adjuvant radiation is effective with good locoregional control. Systemic recurrences are common and may benefit from intensification strategies.

Next-generation sequencing (NGS) for the detection of somatic variants has become the method of choice in a variety of molecular oncology fields and in the clinic. Its use ranges from sequencing entire tumor genomes and transcriptomes to targeted clinical diagnostic gene panels. The NYU Langone Genome PACT (Profiling of Actionable Cancer Targets, LG-PACT) assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing (NGS) of formalin-fixed paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients to detect gene alterations in a targeted panel covering 606 genes and the TERT promoter. Indications for testing are cancer (solid tumors and hematological malignancies) where a mutational profile from multiple genes would be informative for disease stratification, prognosis, or treatment options including targeted therapies and eligibility for clinical trials. The test is intended to provide information on somatic mutations including point mutations, small insertions/deletions (indels), and copy number aberrations for diagnostic and treatment decisions. LG-PACT is a United States Food and Drug Administration (FDA) cleared diagnostic test (510K: K202304). The clinical interpretation of sequencing data of molecular tumor markers from NGS encompasses automated variant calling tools with human interpretation. This final mostly manual review of data step is intensive, involving highly trained scientists, encompassing literature review, interpretation and clinical tier classification by pathologists, who then provide a complete molecular diagnostic report to the treating oncologists. We provide analysis of 1339 clinical genomic profiles from 31 different cancers and their subtypes, comprising of central nervous system (CNS) 792 (59%) cases (incl. meningioma, glioma and glioblastoma), with 267 (20%) cases predominantly of lung, pancreatic and colorectal and 280 of others (21%). Here, we present the technical challenges of validating an NGS oncological diagnostic targeted assay for clinical grade accuracy and sensitivity for patient care. We show how copy number alterations provide a more comprehensive description of the tumors genomic profile. We then outline the utility of targeted panel sequencing based on certified pathologist selection of reportable variants for our current patient cohort. Where analysis of variant detection has led to 49.4% (661/1339) of our clinical tumor samples containing mutations in known therapy targeted genes, 35.6% (477/1339) with mutation detected in other genes, and 15% (201/1339) cases being negative.

Myeloproliferative neoplasms (MPN) are chronic hematologic malignancies characterized by clonal myeloid expansion, inflammation, oxidative stress, and progressive bone marrow (BM) remodeling that may culminate in fibrosis and secondary acute leukemia. Here, we evaluated the therapeutic efficacy and the underlying mechanisms of melatonin (MT) and liposomal melatonin (nano-MT) in preclinical MPN models. MT selectively inhibited clonogenic growth of patient-derived peripheral blood mononuclear cells and induced pluripotent stem cell-derived CD34 hematopoietic stem and progenitor cells in comparison to healthy controls. This effect was associated with increased apoptosis, reduced reactive oxygen species (ROS), and decreased glucose uptake, independently of MT receptor signaling. Transcriptomic profiling of primary MPN CD34 cells revealed suppression of MYC targets, G2M checkpoint signaling, ROS, and glycolysis pathways. In co-culture models, MT reduced stromal -smooth muscle actin and phosphorylated SMAD2/3, indicating inhibition of TGF-{beta}-driven mesenchymal stromal cell-to-myofibroblast formation. In tamoxifen-inducible SclCreER;JAK2V617F mice, nano-MT achieved efficient spleen and BM targeting. Therapeutically, nano-MT reduced erythrocytosis, myeloid progenitor expansion, and BM IL-1{beta} levels. Longitudinal micro-computed tomography and histological analyses demonstrated normalization of BM architecture, reduced osteosclerotic remodeling and splenomegaly, decreased reticulin deposition and megakaryocyte numbers. In a dose-escalation study, nano-MT restored erythrocyte, hematocrit, and platelet counts and normalized megakaryocyte-erythroid progenitors. Combination treatment with ruxolitinib further reduced leukocytosis, neutrophilia, and monocytosis. Collectively, these findings demonstrate that (nano-)MT attenuates MPN and BM remodeling by targeting metabolic, inflammatory, and fibrotic pathways. This study provides the first evidence for a therapeutic benefit of nano-MT in MPN and establishes a rationale for further translational evaluation.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by high metastatic dissemination, therapy resistance, and poor clinical outcome. Inhibitor of differentiation 1 or ID1, is frequently overexpressed in PDAC and is associated with tumour progression and adverse clinical outcome. However, the mechanisms governing its post-transcriptional regulation remain insufficiently characterized. Here, we identify tumour-suppressive miR-615-5p as a regulator of ID1 expression in PDAC. Integrative in-silico target prediction prioritized miR-615-5p based on seed complementarity and thermodynamic stability with the ID1 3' -UTR. Expression analysis of available PDAC clinical datasets revealed reduced miR-615-5p expression associated with increased ID1 expression. Direct association was validated using luciferase reporter assays, where miR-615-5p suppressed 3' -UTR reporter activity of ID1 in a sequence dependent manner, while mutation of the predicted binding site attenuated this effect. Further biotinylated-RIP and AGO2-RIP assays demonstrated the co-enrichment of ID1 transcripts and miR-615-5p with AGO2 associated RISC complexes, while AntimiR mediated inhibition of miR-615-5p perturbs association between miR/ID1 to AGO2, supporting interaction specificity. Functionally, modulation of miR-615-5p altered ID1 expression and impacted PDAC cell migration in vitro. Mechanistic analyses further indicated that the miR-615-5p/ID1 axis influences autophagic flux where miR-615-5p mediated inhibition of autophagy suppresses ID1 dependent cellular migration. Collectively, these findings define a previously uncharacterized miRNA-dependent regulation of ID1 expression and link this axis to autophagy-associated migratory responses in PDAC cells. The study expands the post-transcriptional regulatory landscape of ID1 and provides a possible mechanism where suppression of miR-615-5p leads to ID1 overexpression and subsequent poor clinical outcome in PDAC cells.

Liver metastases (LM) remain a major cause of death from different cancer types, in particular malignancies of the gastrointestinal tract. Liver metastases predict a poor response to immunotherapy due, among others, to the immunotolerant microenvironment (ME) of the liver and loss of local and systemic cytotoxic T cells. Thus, strategies that can reprogram the immune ME of the liver and restore cytotoxic T cell reactivity are being sought. We previously reported that estrogen signaling blockade impedes the growth of LM by reducing MDSC accumulation and monocyte/macrophage polarization. The aim of this study was to elucidate the underlying mechanism(s) and assess whether estrogen signaling in the myeloid lineage was driving the immunotolerant ME of LM. To this end, we generated mice with conditional myeloid cell-specific deletions of estrogen receptors (ER) or ER{beta} and analyzed in these mice the effect of ER loss on the liver immune ME and the outgrowth of LM. In mice with ER, but not with ER{beta} deletion, we observed a marked reduction in the growth of murine colon carcinoma MC-38 liver metastases as compared to their respective controls. Flow cytometry and immunohistochemistry revealed a decrease in macrophages that were polarized to the pro-tumorigenic M2-like phenotype and a concomitant increase in activated CD8+ T and NK cells relative to controls. Bulk RNAseq analysis performed on hepatic immune cells infiltrating the liver revealed changes in the expression of key cytokines/chemokines mediating immune cell recruitment, activation and polarization, including Ccl5 (upregulated) and Csf1 (downregulated). Taken together, the data suggest that ER signaling in myeloid-derived cells programs the immune landscape and contributes to an immunosuppressive and metastases-growth permissive ME in the liver.

Ethnopharmacological relevance: Australian Indigenous medicinal plants represent a valuable yet underexplored source of bioactive compounds with potential therapeutic relevance. The Iningai community of Central Queensland has traditionally used native plants to manage conditions associated with inflammation, pain, infection, and general illness. Scientific evaluation of these plants may provide evidence for their customary applications and identify bioactivities relevant to anticancer biodiscovery. Aim of the study: This study evaluated leaf and stem extracts of seven medicinal plants-Pittosporum angustifolium, Alphitonia excelsa, Calytrix microcoma, Geijera parviflora, Melaleuca uncinata, Gossypium australe, and Eucalyptus similis-traditionally used by the Iningai community, focusing on three biological processes relevant to cancer: oxidative stress, inflammation, and cellular proliferation. Materials and methods: Antioxidant activity was assessed using DPPH radical-scavenging and ferric reducing antioxidant power (FRAP) assays. Anti-inflammatory activity was evaluated in lipopolysaccharide (LPS)-stimulated THP-1 macrophage-like cells by profiling IFN-, TNF-, IL-6, IL-12, IL-18, and IL-23. Antiproliferative activity was assessed using MTT-based viability assays in human and murine liver cancer cell lines (Huh7, Hep3B, Hep-55.1c, and A52). Results: The extracts exhibited distinct biological activity profiles. G. parviflora stem and C. microcoma leaf extracts showed the strongest antioxidant activities, whereas P. angustifolium stem exhibited the weakest radical-scavenging capacity. Cytokine responses were extract-specific, with E. similis leaf extract demonstrating broad and pronounced suppression of multiple LPS-induced pro-inflammatory cytokines. Several extracts produced concentration-dependent reductions in liver cancer cell viability, with P. angustifolium stem exhibiting the most consistent and potent antiproliferative activity across the cell lines tested. Notably, strong antioxidant or anti-inflammatory activity did not necessarily correspond with antiproliferative activity. Conclusion: Australian Indigenous medicinal plant extracts demonstrated distinct antioxidant, immunomodulatory, and antiproliferative activities rather than uniform bioactivity across experimental systems. The divergent activities of G. parviflora, C. microcoma, E. similis, and P. angustifolium highlight the importance of integrated biological screening and support the value of Indigenous knowledge-guided biodiscovery. These plants represent promising sources for further investigation of selective bioactive compounds with potential relevance to anticancer drug discovery.

Objective White smoke inhalation injury (WSI) causes severe acute lung damage with no specific therapy currently available. Sphingolipid metabolism is implicated in pulmonary inflammation, but its transcriptional regulatory landscape in WSI remains unexplored. This study aimed to identify key sphingolipid metabolism related genes and evaluate their regulatory roles and therapeutic potential in WSI. Methods We established a rat model of WSI and performed integrated bulk RNA sequencing, weighted gene coexpression network analysis (WGCNA), and single-cell RNA sequencing (scRNAseq) to screen for differentially expressed sphingolipid metabolism-related genes (DESRGs). Protein-protein interaction (PPI) network with four centrality algorithms was used to prioritize hub genes. In silico gene knockout and molecular docking were conducted to assess regulatory functions and identify potential drug candidates. Results We identified 22 DESRGs that were predominantly enriched in DNA replication and cell cycle pathways rather than canonical sphingolipid metabolic processes. PPI consensus prioritized three hub genes--Top2a, Ttk, and Ccna2--with Top2a exhibiting the highest expression in epithelial cells and significant downregulation after smoke exposure. ScRNAseq revealed immune cell infiltration and epithelial differentiation trajectories. Virtual knockout showed that Top2a depletion affected the largest transcriptomic fraction (~0.4%) and was enriched in lysosome biogenesis, innate immunity, phagocytosis, and lipid catabolism. Molecular docking identified thalidomide as a high affinity ligand for Top2a (Vina score: -8.5 kcal/mol). Conclusion Our multiomics integrative framework identifies Top2a as a central regulatory hub linking sphingolipid associated inflammation to epithelial responses in WSI, and nominates thalidomide as a potential drug repurposing candidate. These findings provide prioritized targets for future translational investigation.

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown great promise in treating bladder cancer and are now part of the standard treatment for advanced disease. However, many patients still fail to respond to treatment and at present many biomarkers are assessed but have yet shown only limited results. Therefore, with the advent of combination treatments and the increase of immune related adverse event, the search for reliable predictive biomarkers is paramount. Using a multiplexed enhanced FRET-FLIM based technique (QF-Pro) we quantified the interaction of PD-1/PD-L1, CTLA-4/CD80 and TIGIT/CD155 immune checkpoints in a pre-treatment TMA of 46 patients treated with atezolizumab. The association between higher PD-1/PD-L1 ICP interaction state and treatment efficacy was demonstrated in the male sample cohort, where it identified patients with better PFS. Conversely, patients exhibiting higher CTLA-4/CD80 engagement had a worse response to atezolizumab. Remarkably, the dual assessment of patients with high PD-1/PD-L1 and low CTLA-4/CD80 allowed to identify the best responders. These results indicate that the monitoring of patients immune profile in urothelial carcinoma might be critical in identifying patients who may benefit from combination therapy.

Despite the success of chimeric antigen receptor (CAR) T cell therapy for treatment of B cell acute lymphoblastic leukemia (B-ALL), its translation to acute myeloid leukemia (AML) has been hindered by limited efficacy and significant toxicity. Interferon-gamma (IFN{gamma}) blockade with emapalumab has recently emerged as a promising strategy to mitigate CAR T cell-related toxicities in B cell malignancies, based on evidence that IFN{gamma} is largely dispensable for optimal CAR T cell activity in B-ALL. Whether IFN{gamma} signaling is similarly non-essential in the AML context remains unclear. Here, we demonstrate that disruption of the IFN{gamma} axis impedes anti-AML CAR T cell function and prevents upregulation of target antigen CD123, the apoptotic mediator Fas, and the adhesion molecule ICAM-1 on AML cells. Conversely, exogenous IFN{gamma} enhances CAR T cell cytotoxicity and increases CAR T cell avidity for AML targets. These findings identify IFN{gamma} as a critical mediator of CAR T cell efficacy against AML by promoting increased target antigen expression, enhanced cytotoxicity, and stable CAR T/tumor interactions. Our results suggest that therapeutic IFN{gamma} blockade, including with emapalumab, may compromise CAR T cell responses in AML and should be approached with caution in this disease context.

Patients with history of neck radiation for Human Papilloma Virus (HPV)-associated head and neck cancer experience rapid progression of carotid artery stenosis. The present study sought to determine whether screening and treating asymptomatic carotid artery stenosis in patients with a history of neck radiation is cost-effective. This study is a cost-utility analysis using a probabilistic Markov model over a thirty-year time horizon assessing carotid screening and treatment to avoid neurologic consequences of neck radiation for HPV-associated head and neck cancer. A strategy of no carotid surveillance was associated with a 14.8% cumulative risk of stroke and a strategy of ultrasound surveillance and treatment with TCAR was associated with a 3.0% cumulative risk of stroke. The latter had a median incremental cost of $1.04 million USD and provided a median 39.1 additional QALYs, resulting in a median incremental cost-effectiveness ratio of $26,556 per QALY. In conclusion, this study suggests that ultrasound surveillance and treatment with TCAR for asymptomatic carotid artery stenosis is likely to be cost-effective for patients who have been successfully treated with radiation therapy for HPV-associated head and neck cancer.

Ulcerative colitis (UC) and radiation enteropathy involve intestinal epithelial injury, barrier dysfunction, and inflammation, but effective treatments remain limited. This study evaluated MXC-017, a novel vimentin-targeting urea compound, in mouse models of dextran sulfate sodium (DSS)-induced colitis and radiation-induced enteropathy. Acute colitis was induced in C57BL/6 mice using 3.5% DSS for seven days, followed by regular water for seven days. Radiation enteropathy was induced by 13 Gy total abdominal irradiation. Mice received MXC-017 (150 mg/kg) or vehicle. Disease activity, intestinal permeability, inflammatory and epithelial markers, and histopathology were assessed. MXC-017's effects on cancer stem cell frequency, sphere formation, and migration were also examined in PC-3 and DU-145 prostate cancer cells. MXC-017 reduced DSS-induced colitis severity, accelerated weight recovery, lowered disease activity, partially preserved colon length, and restored barrier function. It also reduced proinflammatory cytokines, macrophage infiltration, epithelial injury, and goblet cell loss while preserving epithelial proliferation and markers of intestinal stem cell function and tight-junction integrity. Following irradiation, MXC-017 improved weight recovery, reduced intestinal permeability, preserved epithelial architecture, and partially mitigated villus shortening. Importantly, MXC-017 did not protect prostate cancer stem cells from radiation. Instead, it reduced stem cell frequency, sphere-forming capacity, and cancer cell migration. These findings support vimentin targeting with MXC-017 as a potential treatment for UC and radiation-induced intestinal toxicity and as an adjunct to radiotherapy for pelvic and abdominal malignancies.

Background: Low-dose computed tomography (LDCT) screening reduces lung cancer mortality but is limited by low uptake and associated with high rates of false-positives and indeterminate-nodules. Breath volatile organic compound (VOC) analysis is a non-invasive candidate biomarker approach that could complement LDCT, but prior work has relied on laboratory-based high-resolution mass spectrometry (HRMS), limiting point-of-care deployment. Methods: In this pilot study, breath samples were collected from 40 patients with treatment-naive, pathologically confirmed non-small cell lung cancer (NSCLC) and 25 lung-cancer-screening-eligible healthy controls. Paired samples were analyzed via a compact point-of-care GC-MS platform (CLARION) and a laboratory HRMS reference. Diagnostic classification models were built independently for each platform using elastic net logistic regression with leave-one-out cross-validation, and performance was evaluated by area under the receiver operating characteristic curve (AUC). Results: CLARION identified 103 VOCs across breath specimens, compared to over 900 identified by HRMS. Despite this difference in panel size, CLARION achieved diagnostic performance nearly identical to HRMS for distinguishing NSCLC cases from controls (AUC 0.864 vs. 0.863). Compared to controls, performance statistics were similar for early-stage NSCLC (AUC 0.854 vs. 0.841) and adenocarcinoma (AUC 0.770 vs. 0.787). VOCs of interest include p-cymene, phenol, propylbenzene, tetradecane, {beta}-ocimene, 2,3-dihydro-indole, and 1-methylthio-(Z)-1-propene. Conclusion: A compact, point-of-care breath GC-MS platform achieved diagnostic performance for NSCLC detection comparable to a laboratory HRMS reference despite a substantially smaller detected VOC panel. These findings support continued development of point-of-care breath VOC testing as a non-invasive, field-deployable complement to LDCT-based lung cancer screening.

Bulk telomere length measured from tumour sequencing is routinely interpreted as a property of the cancer cells. However, a tumour specimen is a mixture, and the patient who supplies it has a telomere length of their own. Here I re-analyse published pan-cancer telomere estimates and ask how much of a tumour's telomere length is patient-specific. A calibration step comes first. Whole-genome and low-pass estimates recover the known cross-sectional attrition of leukocyte telomeres with age, at 26.6 bp per year in blood normals, whereas whole-exome estimates do not. After adjustment for cancer type, sequencing centre and sex, the exome slope is minus 0.6 bp per year. In 684 blood-normal aliquots sequenced by both assays, the whole-genome estimate declines at 38.9 bp per year, whereas the exome estimate from the same DNA shows no detectable decline. The difference between assays is 41.5 bp per year, with P = 3 x 10^-10. Because exome data constitute 78.6% of the original resource, downstream analyses use only whole-genome and low-pass libraries. Within those data, tumour telomere length tracks the patient's matched-normal telomere length. The Spearman correlation is 0.395 in TCGA, with positive associations in 22 of 23 cancer types. This finding replicates in PCAWG using a different telomere estimator, with a correlation of 0.472 and positive associations in all 24 histologies examined. Adjustment for cancer type, sequencing centre and library type leaves a regression coefficient of 0.385. The association is also stable after adjustment for age, sex, tumour purity, leukocyte fraction, ploidy, sequencing coverage and continental ancestry, with coefficients ranging from 0.406 to 0.429. Pure normal-cell admixture is rejected as the sole explanation. Under a two-compartment mixture model, the coefficient for host telomere length is expected to equal 1 and the host-by-purity interaction to equal minus 1. These restrictions are jointly rejected with P = 0.001. Tumour purity, leukocyte fraction and age each explain only about 1 to 3% of within-cohort variance and do not alter the cross-cancer ranking. By contrast, the between-cohort coefficient is not directly interpretable. Its apparent near one-to-one relationship with tissue-associated telomere length depends strongly on which tissue supplies the matched-normal reference and on the statistical spread of that predictor, falling to 0.44 when organ-matched solid tissue is used. Bulk tumour telomere length is therefore a composite phenotype containing a replicated patient-specific component. Telomere biomarker studies should include matched-normal telomere length as a covariate rather than treating tumour telomere length as exclusively tumour-intrinsic.

Hybridoma screening in semi-solid medium typically employs antigens labeled with visible fluorophores (e.g., FITC, AF488) to enable single-step identification of antibody-secreting clones. However, conventional chemical conjugation via NHS-esters or isothiocyanate groups frequently modifies lysine residues located within epitopes, potentially abrogating antibody recognition of these critical regions. Here, we describe a SpyTag SpyCatcher-based site-specific labeling strategy that circumvents epitope damage during semi-solid medium screening. A 16-amino-acid SpyTag was genetically fused to the C-terminus of the target antigen, enabling covalent conjugation to an sfGFP SpyCatcher fluorescent probe. In semi-solid medium supplemented with SpyTag-antigen and sfGFPSpyCatcher, positive hybridoma clones were readily identified by distinct fluorescent halos, whereas negative clones showed no detectable signal. Notably, the site-specific method yielded a significantly higher frequency of fluorescence-positive clones compared to the conventional AF488-labeled antigen method, suggesting that epitope preservation enhances screening recovery. Furthermore, this approach did not impair hybridoma growth or final clone positivity, offering a simple, rapid, and epitope-compatible method for monoclonal antibody screening.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

Chemoradiotherapy (CRT) is the standard-of-care therapy for many solid malignancies, yet predictive biomarkers of treatment response remain limited. We identified a germline single nucleotide polymorphism (SNP) in an intrinsically disordered region of the lysine demethylase KDM3C/JMJD1C (p.S464T) that is associated with CRT outcomes in locally advanced rectal cancers (LARC) and head and neck squamous cell carcinoma (LA-HNSCC). In silico modeling with AlphaFold predicted S464T substitution influenced interaction between phosphorylated KDM3C and RNF8 FHA domain. In cellular models, conversion of S464 to T464 increased sensitivity to DNA-damaging agents. S464T substitution impaired damage-induced MDC1-RAP80 signaling and downstream RAP80-BRCA1 colocalization. SNP carrying cells impaired DNA repair causing genotoxic stress that is associated with increased cGAS-cGAMP innate immune signaling and increased apoptosis. Population analyses with the SNP highlighted an increase incidence of UV-induced skin and other cancers, linking inherited variation in the chromatin regulatory gene KDM3C to genome instability, cancer risk, and therapeutic vulnerability.

Radiosensitizing nanoparticles represent a promising approach for enhancing the efficacy of proton radiotherapy; however, their performance is constrained by restricted penetration into tumor tissue, resulting in preferential perivascular accumulation. Here, we develop a spatially distributed mathematical model of a growing tumor undergoing proton therapy with intravenously administered radiosensitizing nanoparticles to investigate treatment optimization strategies. Using physiologically plausible parameter ranges informed by our own experimental measurements and published data, we demonstrate that co-administration of targeted nanoparticles with antibodies binding to the same tumor receptors can overcome transport-induced localization and promote a more uniform intratumoral redistribution of nanoparticles before irradiation. Population-level simulations across heterogeneous parameter sets suggest that moderate antibody doses consistently prolong tumor regrowth time, whereas higher antibody doses produce a pronounced and robust increase in tumor cure probability under a single high-dose irradiation regimen representative of preclinical settings. A key conceptual result of our analysis is the asymmetric risk associated with antibody co-administration. In contrast to antibody--drug conjugates, for which excessive dosing of unconjugated antibodies may severely compromise therapeutic efficacy, co-administration of antibodies with nanoparticle-based radiosensitizers constitutes a "safe-by-design" strategy with respect to tumor cell kill in the modeled single high-dose irradiation setting: although excessive antibody doses may yield suboptimal outcomes, they cannot reduce tumor cell kill below that achieved with targeted nanoparticles administered without antibodies. These findings identify antibody-mediated spatial redistribution of radiosensitizing nanoparticles as a favorable strategy that is expected to provide robust therapeutic benefit despite substantial variability in tumor characteristics.

The role of UBE2N in myeloid cell-mediated immune suppression in cancer remains undefined. Here, we examined the function of UBE2N in myeloid cell-mediated tumor progression using a temporally inducible myeloid-specific knockout model (LysMCreERUbe2nfl/fl). Temporally induced deletion of Ube2n in myeloid cells (Ube2nMyeKO) significantly hindered growth of YUMM1.7 melanoma. This was accompanied by reduced myeloid cell burden within the tumor microenvironment. We observed altered abundance of PD-1, PD-L1, and SPP1 in the Ube2nMyeKO tumor microenvironment at the tissue level. In vitro analysis showed that knock-in expression of a catalytically deficient UBE2NC87S mutant in bone marrow-derived macrophages (BMDMs) markedly decreased expression of Spp1. We observed decreased SPP1 secretion in Ube2nMyeKO BMDM-conditioned media (CM). Treatment with Ube2nMyeKO BMDM-CM decreased co-expression of PD-1, TIM-3, and LAG-3 on chronically stimulated T cells. Antibody-mediated neutralization of SPP1 in Ube2nWT BMDM-CM decreased PD-1 expression on CD8+ T cells. Together, these findings suggest a role for myeloid UBE2N in YUMM1.7 progression.