Neoplasia

Temozolomide (TMZ) resistance remains a major obstacle in glioblastoma (GBM) therapy, yet the metabolic adaptations underlying this phenotype are incompletely understood. Here, we performed integrative lipidomic, ultrastructural, and pathway analyses to define lipid metabolic reprogramming associated with TMZ resistance and failure of statin-mediated sensitization. Targeted LC-MS lipidomics quantified 322 lipid species across 25 lipid classes in TMZ-sensitive and TMZ-resistant U251 cells under basal conditions and following TMZ, simvastatin, or combination treatment. Multivariate analyses (PCA, PLS-DA, and volcano plots) revealed a robust and treatment-resilient lipidomic signature in resistant cells characterized by enrichment of lysophospholipids, sphingolipids, and cholesteryl esters, alongside depletion of glycerolipid and phospholipid pools. Complementary univariate analysis confirmed these changes at the species level, demonstrating consistent elevation of lysophosphatidylcholine/ethanolamine, glycosphingolipid subclasses, and cholesteryl esters, together with reductions in phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and diacylglycerol intermediates across multiple treatment conditions. In contrast, sensitive cells displayed dynamic lipid remodeling, including phosphatidylinositol and phosphatidylethanolamine enrichment associated with autophagic membrane expansion. KEGG pathway analysis linked the resistant phenotype to Rap1, PI3K-Akt, and phospholipase D signaling networks regulating vesicle trafficking and membrane homeostasis. Transmission electron microscopy confirmed a vesicle-rich intracellular architecture consistent with persistent autophagy flux blockade in resistant cells. Collectively, these findings define a stable lipid metabolic program characterized by lysophospholipid expansion and cholesteryl ester accumulation that supports membrane integrity and therapeutic resistance. Targeting lipid buffering and cholesterol storage pathways may represent a promising strategy to overcome chemoresistance in glioblastoma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/712341v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@178acd7org.highwire.dtl.DTLVardef@19b6a79org.highwire.dtl.DTLVardef@6b3904org.highwire.dtl.DTLVardef@16c3d01_HPS_FORMAT_FIGEXP M_FIG C_FIG Lipidomic and autophagy differences between non-resistant (NR) and temozolomide-resistant (R) glioblastoma cells. NR cells show dynamic lipid remodeling and treatment-dependent autophagy responses, whereas R cells maintain blocked autophagy flux and persistent enrichment of LPC, SM, and cholesteryl esters across treatments.

Boys experience an overall increased incidence of several childhood cancers, including medulloblastoma, a clinically heterogeneous cerebellar tumor. In subtypes of Group 3 and Group 4 medulloblastoma, males are three times more prevalent than females. As medulloblastoma is suspected to initiate during fetal development, we hypothesized that this sex bias reflects a combination of prenatal, sex-specific developmental processes and somatic alterations. To test these hypotheses, we compiled a large multi-omics dataset from children with medulloblastoma, which revealed sex-specific alterations, including frequent loss of the inactive X chromosome in females with Group 4. Generation of a sex-matched single-cell transcriptome atlas of the developing murine cerebellum enabled investigation of putative developmental factors underlying sex bias. Progenitors giving rise to Group 3/4 subgroups were more abundant, more proliferative, and harbored more open chromatin for recruitment of LMX1A and OTX2, master transcription factors defining Group 3/4 identity. Advanced genetically engineered mouse models and human cerebellar organoids were leveraged to determine whether sexual dimorphism arises from intrinsic or extrinsic factors. These models showed that the XY genotype contributed to the phenotype, but the predominant effect was driven by presence of the male gonadal hormone testosterone. Our findings provide a sex-specific genetic and neurodevelopmental explanation for male bias in an aggressive pediatric brain tumor. Outcomes from this study may inform novel treatment strategies delivered according to sex and are likely to be broadly applicable to other sex-biased malignancies arising in early life. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/714163v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@3a06faorg.highwire.dtl.DTLVardef@1a01bb7org.highwire.dtl.DTLVardef@7bc9c2org.highwire.dtl.DTLVardef@fb206d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Engineering macrophages with chimeric antigen receptors is emerging as a promising cancer therapeutic. Chimeric antigen receptor-expressing macrophages (CAR-Ms) engineered to recognize tumor-specific antigens have been shown to inhibit tumor growth and activate adaptive immune responses, leading to robust tumor control in animal studies. Based on this work, clinical trials have been initiated. While the trials have shown promise, challenges remain. The dynamic interactions between CAR-Ms and cancer cells and the exact mechanisms driving anti-tumor effects remain poorly defined. Defining the dynamic interactions between CAR-Ms and cancer cells will provide critical insights for optimizing future CAR-M design and improving therapeutic efficacy. We sought to directly visualize CAR-M interactions with glioblastoma cells at high-resolution and in real-time using CAR-Ms engineered to recognize Neural-Glial Antigen 2 (NG2), an antigen expressed on glioblastoma cells. Using patient-derived glioblastoma cells, we formed glioblastoma spheroids and embedded them in a 3D matrix together with CAR-Ms. Using time-lapse microscopy, as expected, we found that NG2-targeting CAR-Ms engulfed glioblastoma cells. However, excitingly, we found that NG2-targeting CAR-Ms blocked >85% of glioblastoma cell invasion in 3D. This inhibition of glioblastoma invasion was not due to a significant change in CAR-M polarization states. Together, these data suggest that NG2-targeting CAR-Ms both engulf glioblastoma cells and block glioblastoma invasive behavior.

Atypical teratoid rhabdoid tumor (ATRT) is a malignant brain tumor of children that has an overall survival of less than 40 percent even with aggressive therapy. We identified upregulation of the mitogen activated protein (MAP) kinase pathway in ATRT. The novel, brain-penetrant MEK inhibitor mirdametinib inhibited the growth of ATRT cell lines in culture at nanomolar concentrations. Mirdametinib suppressed proliferation as measured by BrdU incorporation and induced apoptosis as measured by cPARP and Annexin V staining. Monotherapy with mirdametinib extended the life of mice bearing orthotopic xenografts. Combination therapy with the brain-penetrant cyclin dependent kinase 4/6 inhibitor abemaciclib further suppressed growth and BrdU incorporation in ATRT cell lines representing all molecular subgroups. Mirdametinib and abemaciclib combined to extend survival of mice bearing orthotopic ATRT xenografts. In conclusion, mirdametinib has single agent activity against ATRT and combines with abemaciclib to decrease proliferation and extend survival in orthotopic xenograft models of ATRT.

Ionizing radiation can be an effective therapy for prostate cancer. Unfortunately, however, more aggressive prostate cancers such as neuroendocrine prostate cancer (NEPC) are often radiation resistant, which contributes to their high degree of morbidity and mortality. In this study, we used an unbiased approach to identify novel mechanisms that contribute to resistance to radiation and that are associated with neuroendocrine differentiation. Specifically, we compared the expression of cell surface proteins by mass spectrometry in prostate cancer cell lines that had been either untreated or treated with radiation to induce resistance, a process that also promotes neuroendocrine differentiation. Among the proteins identified by this screen, we focused on folate receptor (FR) because of its known biological functions and the fact that it is a validated therapeutic target. Our data reveal that FR has a causal role in enabling prostate cancer cells to resist radiation. Importantly, we also demonstrate that the expression of FR is regulated by HIF-1, which also has a causal role in radiation resistance and neuroendocrine differentiation. Given that the ability of cells to resist damage and death in response to ionizing radiation depends largely on their ability to buffer the substantial increase in reactive oxygen species (ROS) that is generated by radiation, we also demonstrate that the folate-FR axis promotes radiation resistance by sustaining intracellular glutathione levels that buffer this increase in ROS. In summary, the data reported here highlight a novel role for FR in resistance to ionizing radiation that is intimately associated with the hypoxic microenvironment of NEPC and the ability of the folate-FRa axis to maintain redox homeostasis.

BackgroundGlioblastoma (GB) is a highly aggressive brain tumor with a median survival of approximately 14 months, primarily due to its ability to infiltrate healthy brain tissue both as single cells and in collectives. A deeper understanding of GB cell motility, both individual and collective, is crucial for developing patient-specific therapies. We aimed to characterize migration in patient-derived GB cells using advanced modeling to identify stratification markers and therapeutic vulnerabilities. MethodsWe developed Single-Cell Behavior Live Imaging (ScBLI), an approach integrating live imaging with computational analysis, applied to 30 GB primary cell cultures. Trajectories and morphological features were tracked and analyzed. Diffusion Entropy Analysis (DEA) was applied to classify trajectories based on the Delta Scaling parameter ({delta} scaling). We evaluated functional responses correlating all findings with clinical outcomes and transcriptomic profiles. ResultsWe analyzed 4,279 cell trajectories. Based on {delta} scaling (range 0.28-0.837), we defined three distinct motility groups: Low (L, {delta} scaling [&le;]0.5), Medium (M, 0.5 < {delta} scaling [&le;] 0.7), and High (H, {delta} scaling >0.7). Functional assays demonstrated that Group H cells are more performant in both positive and negative chemotaxis. Clinically, the three groups showed a clear linear progression with patient survival: High {delta} scaling correlated with the shortest survival (poorer prognosis), while Low {delta} correlated with the longest survival, suggesting that structured motility drives invasiveness. Integrative multi-omic analysis, encompassing both exome and transcriptome profiling, demonstrated that these groups are defined by distinct molecular landscapes rather than poor behavioral traits. Moreover, exome data revealed that Group H is significantly enriched in PTEN alterations (75% vs. 8% in Group L), with PTEN gain-of-function (GoF) mutations exclusively restricted to this group (100% vs 0% in Group L). Notably, within our extended cohort (n=51) currently characterized by whole-exome sequencing, we observed that specific PTEN GoF mutations were associated with a significantly shorter survival compared to PTEN wild-type cases (median OS 6.4 vs 16.6 months; p=0.02), which typically harbor the canonical loss of chromosome 10q. A similar clinical trend was observed when comparing directly GoF carriers to patients with truncating (Ter) alterations (median OS 6.4 vs 14.3 months; p=0.09). Conversely, no survival difference was found between truncating (Ter) mutations and wild-type cases. ConclusionOur findings demonstrate for the first time that migratory efficiency, quantified through DEA, represents a powerful predictor of glioblastoma aggressiveness. Tumor cells adopting highly efficient exploration strategies are strongly associated with poor clinical outcomes and are characterized by distinct molecular signatures, notably PTEN gain-of-function alterations. Statement of significanceOur multi-scale computational framework elucidates emergent behavioral phenotypes as pivotal drivers of glioblastoma progression. By demonstrating a correlation between enhanced migratory efficiency, PTEN gain-of-function, and significantly reduced overall survival, we establish a foundational paradigm for deciphering the emergent complexity governing tumor invasiveness.

Glioblastoma Multiforme (GBM) is a highly aggressive brain cancer characterized by rapid proliferation and extensive remodeling of the extracellular matrix (ECM), leading to progressive tissue stiffening. Although ECM stiffness is known to promote GBM progression, the molecular mechanisms linking mechanical cues to tumor growth remain insufficiently defined. In this study, transcriptomic comparison of GBM tumors and non-neoplastic brain tissue revealed coordinated upregulation of cell cycle regulators and matrisome-associated genes, with survivin (BIRC5) identified as a central node linking proliferative signaling and ECM remodeling networks. Analysis of GBM patient specimens further showed strong nuclear survivin expression in regions with elevated collagen deposition. To directly evaluate stiffness-dependent regulation of survivin, GBM cells were cultured on fibronectin-infused hydrogels with tunable stiffness. Stiff matrices increased survivin expression along with cyclin D1 and cyclin A, consistent with increased cell cycle progression. Pharmacologic inhibition or siRNA-mediated suppression of survivin reduced stiffness-induced proliferation and attenuated expression of matrisome components, including collagens and lysyl oxidase. These findings indicate that survivin functions as a mechanosensitive regulator that coordinates cell cycle progression with ECM production in stiff tumor microenvironments. Collectively, this study identifies survivin as a key mediator linking ECM stiffness to GBM growth and matrisome remodeling. Targeting survivin and its effectors may offer a mechanosensitive strategy to limit GBM growth.

La-related protein 1 (LARP1) is an RNA-binding protein that post-transcriptionally regulates mRNA with potential oncogenic role in multiple cancers; however, its function in endometrial cancer remains unknown. An analysis of the TCGA endometrial cancer cohort showed that overexpression of LARP1 is associated with shorter overall survival (OS) and progression-free interval (PFI) as indicated by Kaplan-Meier analysis. Functional in vitro studies revealed that LARP1 knockdown by two different siRNAs markedly suppressed cell viability and triggered apoptosis, as confirmed by increased protein levels of cleaved PARP1 and cleaved caspase-3. Mechanistically, LARP1 knockdown remarkably reduced E2F1 protein levels as confirmed by immunofluorescence and Western blotting. Clinically, co-overexpression of LARP1 and E2F1 further decreased OS and PFI, suggesting a co-operative oncogenic axis. Importantly, LARP1 knockdown enhanced the sensitivity of ISHI and HEC-1A endometrial cancer cell lines to carboplatin treatment. These findings suggest that LARP1 promotes endometrial cancer survival and resistance to chemotherapy, at least in part, through the regulation of E2F1 and suppression of apoptosis. Targeting LARP1 could represent a promising therapeutic strategy to suppress tumor growth and enhance sensitivity to platinum-based chemotherapy.

The central nervous system (CNS) is a common site of metastatic spread for both non-small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases remain limited. Tyrosine kinase inhibitors have shown promising results in increasing the overall response in brain metastases, owing to their brain penetrance and increased effectiveness; however, their use is limited to the small group of tumors carrying specific oncogenic drivers. Among these, inhibitors with activity against neurotrophic tyrosine receptor kinases (NTRKs) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the brain metastatic niche and can, in a paracrine manner, activate NTRK function in cancer cells. Here we show that NTRKs are expressed in primary tumors, brain metastases, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous BDNF and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro, and effectively prevented brain metastatic colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for brain metastases of WT-NTRK+ lung cancers, and therefore, NTRK inhibitors can be used to target non-fusion NTRK function to prevent or decrease brain metastases. SIGNIFICANCEThese studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

BackgroundGallium (Ga) is a promising anti-tumor agent; however, its precise molecular targets in osteosarcoma remain debated. While current paradigms largely attribute its toxicity to reactive oxygen species (ROS) and ferroptosis, understanding its true mechanism is essential for overcoming therapeutic resistance. This highlights the need for interdisciplinary approaches, such as metabolomics, to unveil novel vulnerabilities in cancer metabolism. MethodsWe employed an interdisciplinary strategy utilizing high-resolution liquid chromatography-mass spectrometry (LC-MS) metabolomics and 13C2-glutamine stable isotope tracing in osteosarcoma cells to elucidate the cytotoxic mechanisms of gallium nitrate. Scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) was utilized for elemental mapping, and in silico modeling was applied to evaluated metal binding dynamics. Furthermore, synergistic effects were tested by combining gallium with the DNA-damaging agent cisplatin. ResultsOur metabolic profiling revealed a profound bifurcation characterized by the systemic depletion of glycolysis and pentose phosphate pathway intermediates, coupled with a novel ribonucleotide accumulation bottleneck. The observed distinct signature strongly implicated ribonucleotide reductase (RNR) as the primary enzymatic target. In silico modeling and SEM-EDS visually and thermodynamically confirmedthat gallium acts as a structural decoy for iron within the RNR active site. The co-localization induces functional iron starvation rather than canonical ferroptosis. Furthermore, isotope tracing confirmed that elevated ROS is a consequence of overall metabolic failure, not the primary driver of cell death. Crucially, gallium functioned as a metabolic DNA repair inhibitor, synergizing potently with cisplatin to prevent the repair of platinum-induced DNA lesions. ConclusionsGallium selectively sensitizes highly proliferative sarcoma cells by disrupting RNR-mediated DNA precursor synthesis, while sparing normal osteoblasts. Leveraging metabolomics to uncover this state of functional iron starvation provides a rational, interdisciplinary framework for developing gallium-based combination therapies designed to break platinum resistance in clinical oncology.

Tyrosine Kinase inhibitors (TKIs) are widely used as effective chemotherapeutic agents for treating patients with EGFR-mutated NSCLC. Unfortunately, after treatment, patients eventually develop resistance to TKI therapy. The most common resistance mechanism for the TKI Osimertinib is the overexpression of the MET Proto-Oncogene, Receptor Tyrosine Kinase (MET). We previously demonstrated that miR-411-5p A-to-I edited at position 5 (miR-411ed) can directly target MET in A549 and H1299 cells. MiR-411ed in combination with Osimertinib reduced cell proliferation in two TKI resistant EGFR-mutated cell lines: HCC827R and PC9R. MiR-411ed did not downregulate MET expression in HCC827R, suggesting an alternative mechanism for TKI response. In this study, we aim to identify the mechanism of miR-411ed TKI response using a multi-omics approach of RNAseq and protein mass spectrometry. In our cellular model, we identified miR-411ed affected genes independent of MET activity, resulting in 211 genes (RNAseq) and 36 proteins (proteomics). Pathway analysis identified an increase in interferon signaling for RNAseq and combined omics, and a decrease in ERK/MAPK signaling in proteomics. Using the IsoTar target prediction tool, we identified STAT3 as a key regulator and confirmed STAT3 protein downregulation upon transfection with miR-411ed. We further investigated the effect of miR-411ed in vivo, observing a reduction in tumor size with miR-411ed in combination with Osimertinib but not with miR-411ed or Osimertinib treatment alone, confirming the effectiveness of miR-411ed in TKI response.

As potent triggers of innate immunity, STING agonists hold promise as active immunotherapeutic agents for cancer treatment. Second-generation STING agonists, suitable for systemic delivery, are being investigated in preclinical research and have entered clinical trials. Here, the novel synthetic STING agonist, BI-1703880 (STINGa), which was designed for intravenous delivery, was investigated for anti-tumour and immunological effects. We show that STINGa activates the STING pathway and results in a transient and dose-dependent upregulation and secretion of interferons and proinflammatory cytokines in vitro and in vivo. We show that intravenous administration of repeated dosing with low-dose STINGa is well tolerated. We report that radiotherapy (RT) and STING agonism synergizes to generate innate immune cell and CD8+ T cell responses that control tumour growth. Anti-tumour activity induced by combined RT / STINGa was reduced in mice lacking a functional immune system. RT / STINGa combination treatment also initiated development of protective immune memory. RT / STINGa upregulated PD-L1, PD-1 and CTLA-4 in the tumour microenvironment. Our findings show that combining RT / STINGa with immune checkpoint inhibitors further increases therapeutic benefit. Our data confirm STING as a therapeutic target in cancer and support the clinical development of BI-1703880 STING agonist, thereby suggesting radiotherapy as a potential combination for enhancing anti-tumour efficacy.

Diffuse midline gliomas (DMGs) are a deadly class of pediatric high-grade brain cancers. Approximately 80% of pontine DMGs feature a dominant, somatic, heterozygous point mutation in the non-canonical histone H3.3-coding gene H3-3A. This dominant-negative mutation replaces lysine 27 with methionine (K27M) and prevents global K27 di- and tri-methylation of all wild-type histone H3 proteins. We aimed to target the H3.3K27M onco-histone pre-mRNA with splice-switching antisense oligonucleotides (ASOs) designed to promote skipping of H3-3A exon 2, as this constitutive exon comprises both the K27M mutation and the natural in-frame start codon of the gene. The lead ASO identified in a systematic screen specifically induced H3-3A exon 2 skipping, did not affect expression or splicing of the paralog gene H3-3B--which also encodes histone H3.3--and restored global H3K27me3 marks in patient-derived DMG cells grown as neurospheres. In a patient-derived orthotopic xenograft tumor mouse model, the lead ASO reduced proliferation and extended survival. Our results show the potential of exon-skipping ASOs targeting H3-3A exon 2 as a therapeutic option for H3.3K27M-altered DMG. More generally, they exemplify the strategy of using ASOs to induce skipping of a constitutive exon to effectively achieve gene downregulation.

B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in hepatocellular carcinoma (HCC) and represents a promising therapeutic target. However, its roles in tumor cell adhesion, metastatic behavior and immune evasion--particularly in interactions with natural killer (NK) cells--remain incompletely understood. In the present study, B7-H3 was depleted using small interfering RNA and CRISPR/Cas9 in epithelial (Huh7 and HepG2) and mesenchymal (SNU449) HCC cell lines. Tumor cell survival, apoptosis, adhesion, migration and invasion were evaluated using functional assays. Expression of adhesion molecules and immune checkpoint proteins was assessed by flow cytometry and western blotting. Oncogenic signaling pathways were analyzed by examining phosphorylation of Akt, ERK, FAK and STAT3. NK cell-mediated cytotoxicity was assessed using primary human NK cells. B7-H3 depletion reduced clonogenic survival and increased apoptosis in mesenchymal HCC cells under anchorage-independent conditions. Loss of B7-H3 impaired cell adhesion, migration and invasion, accompanied by downregulation of PTGFRN, E-cadherin, integrin 3 and integrin V, and reduced cell-to-cell aggregation under anchorage-independent conditions. B7-H3 depletion also decreased surface expression of PD-L1, PD-L2 and CD47. Notably, B7-H3-deficient cells exhibited enhanced susceptibility to primary NK cell-mediated cytotoxicity. Mechanistically, B7-H3 promoted tumorigenic signaling through Akt/S6, MVP/ERK and FAK/Src pathways in epithelial cells, and through FAK/Src and JAK2/STAT3 pathways in mesenchymal cells. Together, these findings reveal previously unrecognized roles for B7-H3 in coordinating adhesion and NK immune evasion in HCC, and support its therapeutic targeting for next-generation immunotherapies.

BackgroundTherapeutic agents targeting the PD1-PDL1 interaction are of great clinical value, however accurately predicting which patients are most likely to benefit is challenging. Improved predictive biomarkers for anti-PD1 therapy are clearly needed. Quantifying PD1 saturation by PDL1 in tumor tissue has the potential to serve as such a biomarker. Here we report a novel bioassay called the PD1 Ligand Receptor Complex Aptamer (LIRECAP) assay and demonstrate it can be used to quantify the saturation of PD1 by PDL1 in formalin-fixed paraffin-embedded tumor biospecimens. ResultsThe PD1 LIRECAP assay was developed by identifying a pair of RNA aptamers. One aptamer preferentially binds to unoccupied PD1 (P aptamer) and the other to the PD1-PDL1 complex (C aptamer). P and C aptamers were added together to a formalin-fixed sample, and bound aptamer extracted. A 2-color qRT-PCR assay using a single set of primers was used to determine the ratio of the sample-bound C to P aptamers (C:P ratio) which reflected PD1 saturation by PDL1 in the sample. Quantification of PD1 saturation by PDL1 as determined by the PD1 LIRECAP assay correlated closely with PD1-mediated signaling and PD1-PDL1 proximity. Analysis of sarcoma FFPE biospecimens confirmed the assay is technically reproducible on clinical biospecimens. There were significant differences in PD1 saturation by PDL1 between patients as well as considerable intratumoral heterogeneity. ConclusionsThe PD1 LIRECAP assay is novel assay that can be used to quantify PD1 saturation by PDL1 in clinical biospecimens. The assay is technically feasible, reproducible, and has the potential to serve as a superior predictive biomarker for PD1/PDL1-based therapy. Similar assays based on this platform could be used in other systems and settings to quantify interaction between two molecules.

BackgroundMaternal protein restriction results in a 28% reduction in nephrogenic cells and nephron units in rodent offspring by the 17th day of gestation compared to adequate protein intake. AimsThe present study investigates the association between growth factor expression and some developmental pathways that contribute to nephron reduction during embryonic and fetal development. Experimental DesignPregnant C57BL/6-Tg and C57BL/6J mice were assigned to either normal protein intake (NP-17%) or low protein intake (LP-6%) groups. Body weight of male offspring and kidney growth factor expression were assessed on gestation days (GD) 14 and 18. ResultsOn GD 14, LP pups exhibited a 4% higher body mass (0.1035 g) compared to NP pups (0.0995 g, p = 0.005). By GD 18, LP pups demonstrated a 4% decrease in body mass (0.939 g, p = 0.03) and a 10% increase in the number of cells per metanephric cap area. Three genes (Csf2, Il1b, Il2) were downregulated, while seven genes (Bmp2, Csf3, Fgf8, Gdnf, Bmp7, Fgf3, Ntf3) were upregulated. By GD 14, phagophores and autophagosomes in the ureteric bud increased by 197%, with further increases observed by GD 18. Bcl-2 expression increased significantly in ureteric bud cells, and mTOR activity was elevated by GD 18. ConclusionEarly gestational protein restriction modifies renal growth factor gene expression, influencing cell proliferation and autophagy, and may contribute to reduced nephron numbers by the 18th day of gestation. HIGHLIGHTSO_LIThis study examines the effects of a low-protein diet during pregnancy in mice and demonstrates a significant reduction in embryo-fetal body weight between gestational days 14 and 18. C_LIO_LIProtein restriction induces a distinct cellular pattern in the mesonephros, with a 21% increase in CAP cells at gestational day 14 (GD14), followed by a decrease by gestational day 18 (GD18) compared to offspring from mothers on a normal protein diet. C_LIO_LIAdditionally, increased expression levels of key growth factors essential for kidney development were observed at GD 14, comparing LP with NP intake during pregnancy. C_LIO_LISeven genes were upregulated (Gdnf, Bmp2, Bmp7, Tgf, Fgf8, Fgf3, Csf3, Ntf3), while three genes were downregulated (Csf2, Il1b, Il2). C_LIO_LIOverall, these findings indicate that gene regulation, autophagy, and mTOR signaling mechanisms significantly influence nephron numbers in response to gestational protein restriction beyond the 18th day of gestation. C_LI

Importance: Glioblastoma (GBM) cells integrate into neuronal circuits, and preclinical work implicates multiple neurotransmitter (NT) networks as key drivers of invasion and treatment resistance. Whether the integration of GBM within NT-defined large-scale brain networks conveys prognostic information for overall survival (OS) is unknown. Objective: To determine whether NT-specific network involvement of GBM is associated with OS in patients with newly diagnosed Isocitrate dehydrogenase (IDH)-wildtype(wt) GBM. Design, Setting, and Participants: In this observational multicenter cohort study, we analyzed two independent cohorts of adults with histopathologically confirmed IDH-wt GBM. Cohort 1 included 153 patients treated at the University Medical Center Hamburg-Eppendorf, Germany (2012-2024), and cohort 2 comprised 264 patients from the University of Pennsylvania Health System, USA (2006-2018). Preoperative contrast-enhanced MRI was used to derive individual tumor masks, which were spatially mapped onto normative NT-informed structural connectomes spanning 19 receptor and transporter systems. Exposures: Preoperative contrast-enhancing GBM lesions, quantified as patient-specific involvement scores (0-1) within each NT-defined brain network. Statistics: We used partial least-squares regression for variable selection and multivariable Cox proportional-hazards models alongside regularized logistic regression with out-of-sample prediction, adjusted for age, methylguanine methyltransferase (MGMT) promoter methylation, and extent of resection, to test associations between NT-specific GBM network involvement and OS. Results: Across 417 patients in two cohorts, greater GBM involvement within cholinergic networks, defined by normative vesicular acetylcholine transporter (VAChT)-weighted as well as dopaminergic D2 receptor involvement, was consistently associated with reduced OS, independent of age, MGMT status, and resection extent. Further, cholinergic network involvement showed the strongest contribution to the prediction models. Other NT networks did not show reproducible prognostic effects across cohorts. Tumor-intrinsic hypomethylation of acetylcholine receptor-associated regions correlated with imaging-based cholinergic network involvement and mirrored its prognostic relevance. Conclusion and Relevance: Tumor integration into neurotransmitter-specific brain networks is an independent predictor of poorer survival in GBM. By combining routine clinical MRI with normative NT-informed connectome data, this approach delineates a novel systems-level marker of tumor aggressiveness and supports cholinergic inhibition as a putative therapeutic target in GBM.

Colonic adenocarcinoma (COAD) is a major cause of cancer-related mortality worldwide. Various tumors are linked to metastasis-associated in colon cancer 1 (MACC1). This study aimed to analyze public datasets to examine MACC1 expression, signaling pathways, copy number variations, and associations with immune cell subsets in COAD employing bioinformatics. MACC1 expression was elevated in COAD, especially in Wnt signaling and chromatin modifier pathways. Analysis of somatic copy number alterations in The Cancer Genome Atlas-COAD dataset revealed a link between MACC1 and DNA damage repair. MACC1 also showed a negative correlation with genes involved in immune cell infiltration in patients with COAD, including cluster of differentiation (CD)8+ T cells, activated dendritic cells, CD8 T cells, and cytotoxic cells. Collectively, these findings suggest MACC1 as a potential prognostic biomarker and therapeutic target for COAD.

Glioma is the most common primary malignant tumor of the brain, and accumulating evidence indicates that neuronal activity plays a pivotal role in tumor progression. In this study, neuronal activity is modulated in vitro using potassium chloride (KCl)-induced depolarization and midazolam (MDZ)-mediated suppression. MDZ is a neuronal activity modulation medication, commonly used for sedation, anxiolysis, and amnesia in clinics. After treatment, conditioned media derived from these neuronal cultures are subsequently co-cultured with glioma cells. EdU incorporation assays demonstrate that MDZ significantly inhibits glioma cell proliferation in vitro. Furthermore, an orthotopic xenograft glioma model is established to assess the anti-tumor efficacy of MDZ in vivo, as evaluated by tumor volume and Ki-67 immunostaining. Mechanistically, insulin-like growth factor 1 (IGF1) is identified as the neuronal-activity-regulated factor that promotes glioma growth through activation of the PI3K/AKT signaling pathway. Moreover, transcriptomic profiling of brain tissues reveals that MDZ attenuates neuronal activity and downregulates neuron-derived growth factors in both glioma and non-tumor regions, thereby exerting anti-tumor effects in vivo. Collectively, these findings demonstrate that MDZ suppresses glioma progression by suppressing neuronal activity and inhibiting neuron-derived trophic factors, providing new insights into the development of therapeutic strategies for glioma.

BackgroundThe tumor immune microenvironment likely plays a central role in progression of thyroid cancer. As for most other solid tumors, it is unknown if immune dysregulation contributes to earlier, subclinical stages of thyroid tumor development, or whether thyroid tumor heterogeneity might involve differential expression of pro-inflammatory mediators. MethodsThe time course of tumor-associated inflammation was studied in Tg-CreERT2;Braf CA/+ mice representing a model of BRAFV600E-driven papillary thyroid carcinoma (PTC). Tumor growth was estimated by histological examination and magnetic resonance imaging. Cytokine expression was monitored by quantitative RT-PCR, RNAScope and Western blot analyses. ResultsBased on spontaneous BrafCA activation due to leaky Cre activity in a minority of targeted cells tumors developed within a preserved thyroid tissue architecture to multifocal papillary thyroid carcinoma (PTC) over a period of 12 months. Tumorigenesis was accompanied by a gradually increased mRNA and protein expression of interleukin-1beta (IL-1{beta}), interleukin-6 and tumor necrosis factor-alpha (TNF-) starting already before Braf mutant cells commenced neoplastic growth. RNAScope revealed that both follicular cells and stromal cells expressed Il1b whereas Il6 and Tnfa transcripts were mostly confined to neoplastic epithelia. Early cytokine expression was associated with oncogene-induced senescence, whereas during tumor development (3-6 months) and in advanced tumor stages (at 12 months) the cytokine expression pattern differed among glands and tumor foci of the same gland accompanied by a highly variable locoregional lymphocytic infiltration. Oral treatment of mutant mice for 1 month with PLX4720, a vemurafenib prodrug, partially reduced cytokine expression along with inhibited tumor growth and redifferentiation of thyroid function. The magnitude of reduced cytokine expression differed much between glands and among mice of both sexes. ConclusionsThese findings indicate that oncogenic BRAFV600E targeted to the thyroid both stimulates endogenous production of IL-1{beta}, IL-6 and TNF- and recruits inflammatory cells to foci of early tumor development. PTCs of different clonal origin are distinguished by differential expression of pro-inflammatory cytokines. The anti-inflammatory effect of mutant Braf kinase inhibition varies presumably related to heterogeneous tumor development, which evolves from stochastic BrafCA activation suggesting there are clonally different probabilities of acquiring drug resistance among Braf mutant thyroid follicular cells.