Abstract Leptomeningeal metastases (LM) are a severe and fatal complication of advanced HER2-positive breast cancer, with limited effective treatment options. Robust preclinical models are needed to better understand LM biology and to evaluate novel therapeutic strategies. We developed an athymic nude mouse model of LM using human HER2-positive JIMT1-BR3 breast cancer cells selected for leptomeningeal tropism (JIMT1-BR3-LM4). Leptomeningeal tumor progression was evaluated using bioluminescence imaging, magnetic resonance imaging, histopathology, and immunofluorescent analyses. Therapeutic efficacy of two doses of trastuzumab deruxtecan (T-DXd) was compared with trastuzumab, nab-paclitaxel, and trastuzumab plus nab-paclitaxel. Pharmacodynamic effects, drug distribution, lesion burden, and survival were assessed. Bulk RNA sequencing was performed to identify molecular alterations associated with leptomeningeal tropism. T-DXd demonstrated dose-dependent antitumor activity, with the higher dose producing marked reductions in leptomeningeal tumor burden in the brain and spine, as well as decreased lesion number, lesion size, and associated edema. T-DXd significantly reduced tumor cell proliferation and increased apoptosis within leptomeningeal lesions. Drug distribution to leptomeningeal lesions was comparable to, or slightly lower than, trastuzumab. T-DXd reduced both leptomeningeal and limited parenchymal brain metastases and produced similar pharmacodynamic effects in both compartments. Treatment with T-DXd significantly prolonged survival, with a subset of animals exhibiting durable long-term survival. Bulk RNA sequencing identified top pathway alterations, including cytokine–cytokine receptor interactions, calcium signaling, cell adhesion molecules, and TNF signaling. LINC01638, POSTN, and VCAM1 were identified as top differentially expressed genes. Validation of these genes is underway. These findings support clinical evaluation of T-DXd for HER2-positive LM and establish this model as a valuable platform for therapeutic discovery.

The FET family of RNA-binding proteins, FUS, EWSR1, and TAF15, contribute to transcriptional regulation and RNA maturation, but their core functions remain unclear. Chromosomal rearrangements involving FUS, EWSR1, or TAF15 drive multiple cancers, and mutations in the genes encoding the FET proteins are associated with neurodegenerative disease. Here, using nanoscale imaging, we show that endogenous EWSR1 and newly synthesized RNA exhibit a network-like organization with EWSR1 foci forming the nodes of this ribonucleoprotein network. Acute depletion of EWSR1 causes a rapid but transient reduction in nascent RNA levels and cellular metabolic activity without affecting active transcription. Notably, loss of EWSR1 induces a compensatory mechanism involving the reorganization of FUS and TAF15 to closely resemble that of EWSR1, including enhanced clustering with newly synthesized RNA. Together, our findings reveal functional redundancy within the FET protein family that is critical for the homeostatic regulation of nascent RNA levels.

Photobiomodulation (PBM) therapy has been effectively used to relieve pain and inflammation and promote tissue healing and regeneration in a broad range of ailments. Prior work has focused on intracellular mitochondrial cytochrome c oxidase, while extracellular latent TGF-β1 activation had been noted. This work investigated the role of PBM-generated redox signaling and integration in normal oral keratinocytes, using Western blots and pathway-specific small molecule inhibitors. We observed that PBM primarily generates ROS intracellularly within mitochondria, which then diffuse extracellularly to activate latent TGF-β1. This activation triggers ATF-4 expression through both canonical (Smad3) and non-canonical (p38, ERK) TGF-β signaling pathways. We observed a critical role for NFκB as an essential integrator, coordinating these responses as evidenced by the loss of ATF-4 expression following NFκB inhibition (BAY II) after both PBM and TGF-β1 treatments. Proteomic pathway analysis revealed that PBM downregulates inflammatory and apoptotic pathways while activating stress-adaptive responses in the NFκB pathway. A core set of PBM-induced redox, NFκB, and TGF-β signaling targets was identified. These findings suggest that optimal PBM treatment responses require a coordinated action of multiple signaling pathways that optimize cellular adaptation to stress and promote tissue repair rather than protracted inflammation and cell death.

Several cancers arise because of chromosomal rearrangements that result in a fusion oncoprotein which includes the N-terminus of FUS, EWSR1, or TAF15, members of the FET family of RNA-binding proteins. Examples of fusion oncoproteins involving the FET proteins, include FUS::ATF1, FUS::CREB3L1, FUS::DDIT3, EWSR1::CREB1, EWSR1::ERG, EWSR1::FLI1, EWSR1::PATZ1, EWSR1::VEZF1, EWSR1::WT1, TAF15::ZNF384, and TAF15::NR4A3. The FET proteins have similar protein domains, specifically, a N-terminal low complexity domain (LCD), an RNA-recognition motif (RRM), a Zn-finger domain, and a C-terminal nuclear localization signal. Study of the LCDs of the FET proteins has proven essential to determining the biophysical properties of the interactions that define biocondensates, and functional studies have implicated FET proteins as regulators of transcription and RNA maturation. However, the exact functions of the FET proteins remain poorly defined. An enhanced understanding of FET protein function is critical to understanding how the biocondensate properties of these proteins and their functions may contribute to the regulation of gene expression and to tumorigenesis. To investigate the function of the FET proteins, we are using high-resolution microscopy to examine the cellular localization of these proteins at endogenous levels of expression, with an initial focus on EWSR1. To generate endogenous EWSR1 reporter cell lines, we used CRISPR-Cas9 gene editing to introduce a DNA cassette at the 5’ end of EWSR1 exon 1. The DNA cassette expressed a FLAG-FKBP12F36V (FF) peptide that enables the nanoscale imaging of EWSR1 using immunofluorescence (IF) and its depletion via compound-regulated (dTAG-13) proteasomal degradation. Using these modified EWSR1 reporter cell lines, we show that EWSR1 interacts with nascent RNA to form an essential, DNA-independent, non-random ribonucleoprotein network (RNP). Depletion of EWSR1 results in a rapid, but transient reduction in nascent RNA and metabolic activity. Loss of EWSR1 mediates no changes in a marker of transcriptional elongation. However, as EWSR1 is depleted, the expression of FUS and TAF15 increase and concurrently exhibit nuclear reorganization. The observed change in the localizations of FUS and TAF15 reestablishes the formation of non-random RNP networks and restores nascent RNA accumulation. We conclude that the EWSR1 RNP forms an essential scaffold for nascent RNA and in the absence of EWSR1, FUS and TAF15 safeguard this critical function. Soumya Sundara Rajan, Imran Khan, Tamara L. Jones, Langston Lim, Andy Tran, Michael J. Kruhlak, Natasha J. Caplen. Biocondensates in action: The formation and reformation of EWSR1, FUS and TAF15 ribonucleoprotein networks [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr IA004.

Leptomeningeal metastases are devastating complications of advanced HER2+ breast cancer, with limited therapeutic options. We developed a xenograft model of human HER2+ breast cancer cell line JIMT1-BR3-LM4 leptomeningeal colonization by four iterative cycles of intrathecal injection. The model reliably produced leptomeningeal lesions in brain and spinal cord and tumor cells in the CSF, as confirmed by endpoints of BLI, MRI, pathologic analysis and immunofluorescent staining. Upon RNA-seq, the LM model exhibited significant transcriptional changes as compared to the starting brain-tropic line. In a preclinical experiment, two doses of trastuzumab deruxtecan (T-DXd) were compared to human IgG, trastuzumab (T), nab-paclitaxel (nab-P) and T + nab-P for leptomeningeal metastasis. T-DXd demonstrated efficacy in terms of BLI imaging of the brain (P < 0.0001) and spine (P = 0.008), leptomeningeal lesion number and size in the brain (P = 0.06); efficacy was dose-dependent. T-DXd 10 mg/kg reduced leptomeningeal tumor Ki67 positivity (P = 0.0008) and increased apoptosis (P < 0.0001). In the brain, a comparison of leptomeningeal and parenchymal lesion number showed a reduction by T-DXd of 53% and 72%, respectively, compared to human IgG, with comparable effects on tumor proliferation and apoptosis. T-DXd also extended median survival to 38 days compared with 20 -24 days in control IgG or T + nab-P, with some mice surviving beyond 60 days (P = 0.003). These findings support ongoing clinical translation of T-DXd for HER2+ leptomeningeal metastasis and highlight the value of this model for future therapeutic development.

Tuberculosis (TB) remains a leading cause of morbidity and with the highest mortality among infectious diseases (1.3 to 1.5 million deaths per year) worldwide, particularly in high-burden settings. Limitations of current diagnostic tests, and lack of integration across the testing modalities due to a dearth of appropriate computational tools contribute to missed or late diagnoses. This proof-of-concept study aimed to integrate multimodality data obtained from machine learning (ML)-based radiological (High-Resolution Computed Tomography (HRCT) and digital chest X-ray) analyses with immune-biomarker and clinical data employing an automated machine-learning (ML)-based approach (MILO®), with a Segmentation-Guided CT Neural Network (SG-CTNN), to enhance TB diagnosis. Previously developed multiplex serological assays were used for profiling immune-biomarkers as follows: (a) antibodies against ten Mycobacterium tuberculosis (M.tb.) antigens, and (b) seven cytokines, particularly focusing on CXCL10 (IP-10) for its diagnostic value. These biomarker profiles were analyzed by using MILO®. To integrate radiological imaging, microbiology, and blood biomarkers for the detection of TB, a four-stage ensemble approach was employed. Additionally, Random Forest (RF)-based combination of the above diagnostic modalities, along with patient information (symptoms, culture scores), was used to stratify disease morbidity. ML-based HRCT analysis achieved 98.3% sensitivity and 94.8% specificity (internal holdout set) and 98.0% sensitivity in an external database. MILO® models using immune biomarkers (antibodies + IP-10) achieved high sensitivity (100%) and specificity (97.2%) on the generalization set. The final four-stage ensemble approach reached 100% sensitivity. This diagnostic stratification machine learning approach enabled a more accurate identification of severe, moderate, and mild TB cases.

This study describes the design, syntheses, and evaluation of two new near-infrared (NIR) carbocyanine dyes as candidate photosensitizing agents for antimicrobial photodynamic therapy (aPDT). In aPDT, light activates photosensitizers to produce cytotoxic reactive oxygen species (ROS), to remediate treatment-resistant microbial infections. The carbocyanines feature terminal indolium groups connected by a heptamethine chain, enabling absorption of light in the tissue-penetrating NIR region. They also incorporate strategically positioned halogen atoms for improving hydrophobicity, chemical stability, and triplet-state yields. To enhance interactions with negatively charged bacterial cell walls and DNA, the dyes have different net charges at pH 7.0 (+1 vs. +2) and quaternary ammonium contents (QAC = 0 vs. 1). Across all experiments, the di-cationic carbocyanine (QAC = 1) outperformed its mono-cationic counterpart (QAC = 0), showing markedly greater stability in neutral aqueous solutions and generating higher levels of direct plasmid DNA strand breakage and hydroxyl radical/singlet oxygen ROS production under both 780 nm and broad-spectrum (707-759 nm) NIR irradiation. Spectral analyses and competitive binding assays indicated that monomeric and aggregated forms of the di-cationic carbocyanine bind within the minor groove of B-form DNA. At sub-micromolar concentrations, the di-cationic dye exhibited minimal dark toxicity toward cultured E. coli cells. However, upon 780 nm illumination, it was approximately tenfold more effective at inhibiting growth of this Gram-negative bacterium than its mono-cationic counterpart and the established aPDT agents indocyanine green and methylene blue (pH 7.0). These findings highlight the importance of developing di-cationic NIR carbocyanine dyes for aPDT applications.

Background Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) approved for metastatic HER2+ and HER2-low/ultralow breast cancer. It has shown impressive clinical activity for HER2+ brain metastases. We conducted preclinical brain metastasis experiments to understand T-DXd efficacy.Methods Nude mice were intracardially injected with either JIMT1-BR (HER2-2+) or SUM190-BR (HER2-3+) brain-tropic breast cancer cells and dosed with 3 or 10 mg/kg T-DXd or 10 mg/kg control-ADC, with endpoints of metastasis number and size, in both the metastasis prevention and treatment of established disease settings.Results In the JIMT1-BR model, T-DXd at both doses reduced metastasis number by 48% to 88% and size by 32% to 88%; a reduction of HER2 expression by lesions remaining at the experimental endpoint and heterogeneous T-DXd distribution were observed. A distinct dose effect was observed in SUM190-BR with the 3 mg/kg dose inhibiting size and number by 24% to 39% and 10 mg/kg by 72% to 79%; HER2 expression was maintained together with heterogeneous T-DXd distribution. In both models widespread reduced tumor Ki-67 was observed, while increased cleaved caspase-3 primarily costained with T-DXd. We used an in vitro model of the blood-brain and blood-tumor barriers (BBB/BTB) to ask how T-DXd crossed. Data demonstrated T-DXd endocytosis and transcytosis of brain endothelial cells partially reliant on the neonatal Fc receptor (FcRn). BTB transcytosis was accompanied by increased endothelial RAB11FIP5 expression in vitro and in vivo.Conclusions The data confirm T-DXd activity in HER2+ brain metastases and identify important correlates, including heterogeneous uptake, variable HER2 expression at endpoint, tumor cell cytotoxicity, decreased proliferation, and BTB transcytosis.

Brain metastasis is a common and serious complication of metastatic triple-negative breast cancer (TNBC) with few effective treatments. Here, we evaluated the effect of targeting the brain tumor microenvironment via the myeloid colony-stimulating factor-1 receptor (CSF-1R) pathway using the small molecule inhibitor BLZ945. Studies were conducted in two TNBC hematogenous brain-tropic models, 4T1-BR5 and 231-BR, with endpoints of prevention of brain metastasis formation and treatment of established brain metastasis. BLZ945 reduced the formation of brain metastases in both models by 57–65

The number of mobile users on onboard vehicles is increasing continuously. Vehicular users suffer from vehicle penetration loss, fast variation of channel conditions, and signal strengths. These challenges are made even worse by a growing need for high data rates and high quality of service requirements in vehicular scenarios, which cause frequent handovers, call drops, and complete outages. The achievable rate supports adapting techniques for communication to dynamically changing conditions, ensuring reliable and efficient transmission of information despite variations. Moving relay (MR) has been proven to be one of the most promising techniques for handling such scenarios over Rayleigh fading channels. However, the performance of MR for non-vehicular users needs to be analyzed over Rician fading channel in terms of achievable rate. This paper proposes a cooperative non-orthogonal multiple access (NOMA) based system over Rician fading for non-vehicular users with half duplex decode and forward protocol-based MRs in downlink communication.We derive the achievable sum rate of the proposed system for non-vehicular users and analyze its performance in terms of power allocation coefficient, transmit signal-to- noise ratio (SNR), and Rician factor. Also, a comparison of the proposed system with a conventional orthogonal multiple access (OMA) system is provided. The simulation results show that the proposed cooperative NOMA system achieves 11.96 % higher achievable sum rate at 5 dB SNR and 14.88% higher achievable sum rate at 25 dB SNR compared to conventional OMA system.The analytical results are verified through Monte Carlo simulations. The proposed system may be useful for designing high-speed vehicular scenarios in 5G and beyond communication.

Growth factor proteins are essential reagents for cell culture and tissue engineering but require quality control for bioactivity. Currently, growth factor bioactivity measurements made by immunoblot or ELISA are limited in evaluating the kinetics and heterogeneity of cellular responses, and there is a need for convenient methods with higher temporal and spatial resolution. In this study, we evaluate the advantages of using genetically encoded biosensors to quantify growth factor bioactivity in living cells. Using fibroblasts expressing a FRET-based biosensor of ERK activity, we compare methods for quantifying the cellular response across several doses and sources of recombinant basic fibroblast growth factor (bFGF). In contrast to immunological methods, the biosensor-based approach provides single-cell ERK activity kinetics and robust dose-response curves with minimal experimental processing. We additionally demonstrate that this method can assess bFGF activity in induced pluripotent stem cells and resolve spatial activity patterns. We conclude that biosensors represent a rapid, high-quality bioactivity assay extendable to other growth factors and signaling pathways.

Supplemental Fig. 3. ERB-B-2 Status of Patients.

Supplemental Fig. 1. Neurocognitive assessments comparing patients presenting progression versus no progression.

Supplemental Fig. 4. Histogram illustrating ERBB2 relative copy number in plasma and CSF cfDNA samples from 12 patients during their T samples from 12 patients during their Tsamples from 12 patients during their T samples from 12 patients during their T-DM1/TMZ combination therapy.