Theranostics

The limited efficacy of immune checkpoint inhibitor (ICI) therapy in triple-negative breast cancer (TNBC) highlights the need for combination strategies that enhance antitumor responses. Sorafenib, a multikinase inhibitor with anti-angiogenic and immunomodulatory activity, represents a rational partner for ICI-based combination therapy. However, therapeutic responses to such combinations are biologically complex and cannot be fully characterized by any single biomarker or imaging modality. Here, we evaluated ICI therapy combined with sorafenib in the aggressive and ICI-refractory orthotopic 4T1 TNBC model. Therapeutic responses were assessed using a unique longitudinal multimodal imaging framework integrating [Zr]Zr-DFO-anti-CD8 minibody and [{superscript 1}F]FDG PET, as well as perfluorocarbon (PFC)-based {superscript 1}F MRI and hyperpolarized {superscript 1}3C MRS, together with ex vivo analyses. Only the ICI-sorafenib combination suppressed tumor growth, whereas both monotherapies showed limited antitumor activity. Multimodal imaging, together with complementary ex vivo analyses, uncovered coordinated tumor microenvironment (TME) remodeling, including vascular normalization, elevated CD8 cell presence with modest enrichment in the tumor center, delayed increase in phagocyte-associated {superscript 1}F MRI signal coupled with reduced CD206 cell infiltration, and sustained metabolic activity. These findings support ICI-sorafenib combination therapy as a promising therapeutic strategy for TNBC. Therapeutic efficacy reflected coordinated vascular, immune, and metabolic remodeling. This multimodal imaging framework enables non-invasive longitudinal monitoring of these complementary TME changes, providing a comprehensive strategy for treatment assessment in immunotherapy-based combination therapies. One Sentence SummaryLongitudinal multimodal imaging identified a multidimensional TME response signature of effective ICI-sorafenib therapy in TNBC.

Targeted radiopharmaceutical development for ovarian cancer (OC) has been limited by the lack of molecular targets that combine broad tumor expression with minimal normal-tissue distribution. TRA-1-60 (TRA) is a cancer-associated glycoepitope carried by podocalyxin. Here, we evaluated TRA as a target for OC and developed a TRA-directed immunoPET imaging platform. Immunohistochemical analysis demonstrated significantly higher TRA expression in ovarian tumors than in normal adjacent ovarian tissue, with expression maintained across epithelial OC histotypes and disease stages. An engineered anti-TRA single-chain variable fragment-Fc (scFv-Fc) demonstrated robust penetration of three-dimensional tumor spheroids and selective accumulation in intraperitoneal tumors in an immunocompetent syngeneic OC model. Radiolabeling with zirconium-89 generated [Zr]Zr-DFO-anti-TRA scFv-Fc with >98% radiochemical yield. Serial PET/CT imaging demonstrated progressive and sustained radiotracer accumulation at tumor sites through 96 hours, accompanied by declining liver-associated activity and low uptake in most normal tissues. Together, these findings identify TRA as a broadly expressed and accessible tumor-associated glycoepitope and establish TRA-targeted immunoPET as a promising strategy for noninvasive detection of OC. The selective and sustained tumor localization of this platform further provides a foundation for development of TRA-directed radiopharmaceutical therapy, supporting a potential theranostic approach for OC.

IntroductionTherapeutic ultrasound has been extensively studied in ablative and sonodynamic contexts, leaving the intrinsic bioactivity of continuous non-thermal low-intensity ultrasound (LIU) largely uncharacterized. ObjectivesTo characterize the tumor biological and immunomodulatory effects of non-thermal continuous LIU in complementary in vitro and in vivo breast cancer models, underpinned by a standardized exposure platform characterized through finite element simulations and experimental validation. MethodsAcoustic and thermal fields were characterized and optimized using in silico simulations and validated against hydrophone and temperature measurements to ensure homogeneous, non-thermal exposure (1MHz, 1W/cm2, 100% duty cycle). 4T07 murine mammary carcinoma spheroids received 20min LIU treatment, and metabolic activity, apoptosis, and intracellular stress-associated markers were assessed. In a syngeneic orthotopic 4T07 mammary carcinoma model in BALB/c mice, up to six LIU treatment cycles were administered; tumor growth, survival, histopathology, immunohistochemistry, bulk tumor RNA sequencing, spleen volume and plasma cytokine profiles were assessed. ResultsIn vitro and intratumoral temperatures remained within the physiological range ([≤]39{degrees}C) throughout exposure. In spheroids, LIU reduced ATP content by more than 40% and significantly increased apoptotic, Hsp70 and Hsp90 cell fractions. In vivo, cyclic LIU slowed tumor growth, increased intratumoral necrosis, and significantly prolonged time to humane endpoint compared to untreated controls. LIU promoted early intratumoral myeloid cell infiltration and shifted the tumor transcriptome (2,573 differentially expressed genes), with enrichment in gene sets associated with immunogenic cell death, pattern-recognition, inflammatory, and innate and adaptive immune programs and downregulation of pro-tumorigenic pathways. LIU enriched the transcriptional signatures of M1 macrophage polarization and, notably, B-cell compartment engagement, which has not previously been reported for standalone continuous mechanical ultrasound. LIU significantly attenuated tumor-associated splenomegaly and elevated plasma IL-1, TNF-, and IL-10. ConclusionThese results establish a reproducible preclinical platform and provide a hypothesis-generating mechanistic basis for evaluating LIU as an adjunct to immune checkpoint blockade. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/743931v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@31d366org.highwire.dtl.DTLVardef@12df6aborg.highwire.dtl.DTLVardef@9d91adorg.highwire.dtl.DTLVardef@c72b8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Immunotherapy shows limited efficacy in brain tumours, where restricted immune access, antigenic heterogeneity and local immunosuppression constrain durable responses. Low-intensity pulsed ultrasound with microbubbles (LIPU+MB) transiently modulates the blood-brain barrier (BBB) and is widely assumed to enhance immunotherapy by facilitating drug and immune cell penetration into the central nervous system (CNS). However, whether increased anatomical access alone is sufficient to generate effective CNS immunity remains unclear. Here, using a transgenic mouse model with astrocyte-restricted antigen expression, we showed that BBB modulation alone is insufficient to generate functional T-cell immunity in the CNS. Although LIPU+MB enabled rapid T-cell entry, accumulation required prior T-cell activation and integrin-dependent mechanisms, indicating that entry remains governed by canonical immune processes. Moreover, T-cells failed to persist owing to insufficient activation of antigen-presenting cells (APCs) within the CNS. Systemic immune adjuvants (poly-ICLC and IL-2; PI) induced APC activation, promoted tissue-resident-memory-like differentiation and supported durable T-cell responses. LIPU+MB further enhanced these responses by increasing T-cell recruitment, resulting in greater accumulation than with PI alone. Mechanistically, antigen presentation by bone marrow-derived APCs was more critical than that by microglia for the accumulation and persistence of antigen-specifc T-cells in the CNS. In antigenically heterogeneous glioma models resistant to CAR T-cell therapy, combining PI with BBB modulation enhanced the efficacy of immunotherapy, which was mirrored by prolonged survival and endogenous tumour-specific T-cell responses, consistent with epitope spreading. Together, these findings define key limitations of LIPU+MB in enabling effective T-cell therapy and establish that BBB modulation must be coupled to systemic immune activation to support T-cell-mediated antitumour immunity in the CNS.

The immune checkpoint protein PD-L1 plays a pivotal role in tumor immune evasion by binding to PD-1 on immune cells, including T lymphocytes. While the expression and function of PD-L1 have been well studied, the importance of its spatial organization on the cell surface of tumor cells remains poorly understood. In this study, we used super-resolution microscopy combined with biochemical perturbations to investigate the factors regulating PD-L1 clustering and its effects on PD-1 binding and T cell inhibition. We found that PD-L1 is organized into nanoscale clusters at the plasma membrane, with distinct regulatory roles for the actin cytoskeleton, galectin-3, and cholesterol. Disruption of cortical actin increased PD-L1 cluster size, while galectin-3 promoted smaller, denser clusters and increased PD-L1 lateral mobility. Cholesterol depletion reduced PD-L1 cluster size and number and impaired PD-1 binding. These findings indicate that PD-L1 surface organization is collectively regulated by the actin cytoskeleton, galectin-3, and membrane cholesterol within the plasma membrane of tumour cells. Our results provide new insights into the dynamic regulation of PD-L1 and its potential as a therapeutic target in cancer immunotherapy.

Solid tumors are often surrounded by abnormal vasculature and a dense collagen-rich extracellular matrix that severely restrict the infiltration of T cells, including tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR)-T cells. These physical barriers represent a major obstacle to the efficacy of adoptive T cell therapies in solid tumors. We previously identified Formin-like 1 (FMNL1) as a cytoskeletal regulator critical for T cell extravasation and migration through restrictive environments, making it a promising target to improve T cell infiltration into tumors. Here, we developed a bioengineering platform to enhance T cell cytoskeletal dynamics by overexpressing FMNL1 in TILs and CAR-T cells. FMNL1 overexpression significantly increased T cell migration through restrictive pores in transwell assays, supporting enhanced migratory capacity of T cells under mechanically constraining conditions. Importantly, FMNL1 overexpression did not impair T cell reactivation or cytotoxic function in vitro. In murine models of melanoma and lung carcinoma characterized by limited effector T cell infiltration, FMNL1-overexpressing TILs and CAR-T cells had significantly increased accumulation at tumor sites compared to controls. Importantly, enhanced tumor accumulation resulted in improved therapeutic activity, as adoptive transfer of FMNL1-overexpressing CAR-T cells limited tumor growth and prolonged the survival of tumor-bearing mice in multiple melanoma models. Together, our findings identify FMNL1 as a broadly applicable cytoskeletal engineering target to enhance T cell accumulation and persistence in restrictive tumor microenvironments, thereby overcoming a fundamental limitation of adoptive cellular immunotherapy in solid tumors.

The VEGF Receptor-1 (VEGFR1) is a deceptive receptor tyrosine kinase (RTK). In early embryonic development, VEGFR1 negatively regulates angiogenesis by acting like a decoy receptor. Ligand binding transiently phosphorylates the receptor and induces a weak activation, even at high receptor density. Yet, in multiple cancers, overexpression of VEGFR1 plays a central role in tumor vascularization and growth. Unlike many pro-oncogenic RTKs, extensive patient data analysis revealed no somatic mutation in VEGFR1 that may spontaneously activate the tyrosine kinase. The mechanism by which VEGFR1 is activated in cancers has remained an open question for more than two decades. Here, we evaluated the multi-omics profiles of VEGFR1 and its regulators in a pan-cancer database. We observed an inverse correlation between VEGFR1 and PTPRB phosphatase expression in KIRC patients and disease outcome. We observed that patients overexpressing VEGFR1 and deficient in PTPRB expression have a lower likelihood of survival. Using super-resolution single-cell imaging, we discovered that inhibiting PTPRB spontaneously activates VEGFR1 by inducing ligand-independent dimerization, possibly by shifting the equilibrium toward the active state. PTPRB inhibition induces sustained, ligand-dependent phosphorylation of VEGFR1, which may promote tumor vascularization. We conclude that a subtle phosphatase imbalance is fundamental in determining VEGFR1s role in pathological angiogenesis in tumors.

Background: Trophoblast cell surface antigen 2 (TROP2, encoded by TACSTD2) is a transmembrane glycoprotein overexpressed in multiple aggressive epithelial carcinomas. While antibody drug conjugates targeting TROP2 have achieved regulatory approvals, acquired payload resistance and systemic off-target toxicities limit sustained remissions. Radionuclide Drug Conjugates (RDCs) represent a potent alternative modality capable of delivering cytotoxic ionizing radiation directly to target cells. However, selecting the optimal therapeutic radioisotope between long-range beta emitters (177Lu) and short-range, high linear energy transfer (LET) alpha emitters (225Ac) under heterogeneous TROP2 spatial distributions remains an unaddressed clinical challenge. Methods: We developed an automated computational pathology and spatial microdosimetry pipeline to resolve microscopic TROP2 expression gradients and simulate absorbed radiation dose distributions from digitized whole-tissue immunohistochemistry (IHC) sections (N = 14). Optical density matrices were de-convoluted in Hematoxylin-Eosin-DAB (HED) color space to isolate the DAB chromogen. Continuous 2D spatial density distributions and topological surface profiles were reconstructed. Physical radiation energy deposition was modeled using radial dose point kernels for 177Lu (mean range ~670 m, LET 0.2 keV/m) and 225Ac (mean range ~65 m, LET 100 keV/m, 4 alpha particles per decay cascade). Therapeutic Index (TI, ratio of mean target to non-target absorbed dose), target coverage, and spatial specificity were quantified across all specimens. Results: Quantitative image deconvolution revealed that TROP2 expression across the cohort was characteristically focal and clustered, with a mean positive area fraction of 1.55 +/- 2.22% (range: 0.08% to 6.85%) and mean DAB signal intensity of 0.256 +/- 0.043. In all 14 evaluated specimens (100%), 225Ac-labeled RDCs demonstrated superior tumor-to-stroma dose localization compared to 177Lu-labeled RDCs. The cohort-wide mean Therapeutic Index was significantly higher for 225Ac (1.26 +/- 0.14) than for 177Lu (1.01 +/- 0.02, p < 0.0001, paired two-tailed t-test). Because the path length of 177Lu beta particles exceeded target cell nest dimensions by up to 30-fold, 177Lu suffered from severe off-target crossfire spillover into antigen-negative stroma. In contrast, 225Ac confined high-LET ionization tracks strictly within the micro-geographic boundaries of TROP2-expressing clusters. Conclusions: In tumors displaying focal or sparse TROP2 micro-architecture, Targeted Alpha Therapy with 225Ac-RDCs offers a superior biophysical profile over beta-emitting 177Lu-RDCs, maximizing cluster cell kill while sparing adjacent normal tissue stroma. This computational microdosimetry framework provides a practical tool to guide rational isotope pairing in RDC drug design.

Formation of tertiary lymphoid structures (TLS) within the bladder microenvironment because of chronic mucosal inflammation has been associated with variable clinical outcomes. While the immune cell composition and functional states of TLS have been characterized in both non-invasive and muscle-invasive bladder tumors, the TLS-adjacent tumor epithelial compartments remain poorly characterized. Evaluation of a 16-gene TLS signature in treatment-naive tumor bulk RNA sequencing profiles from 283 non-muscle invasive bladder tumors, from patients treated with Bacillus Calmette-Guerin (BCG) immunotherapy, and 348 muscle-invasive bladder tumors from patients treated with immune checkpoint inhibitor therapy revealed overlapping enrichment of immune exhaustion pathways. High TLS gene expression scores correlated with upregulation of immune exhaustion, hypoxia, and epithelial-to-mesenchymal transition (EMT) pathways in tumors from both cohorts. Spatial whole transcriptomic analysis of tumor sections with high TLS density, revealed enrichment of genes associated with EMT, angiogenesis, extracellular matrix remodeling, and B cell receptor signaling pathways in tumor epithelial regions adjacent to TLS, whereas those distant from TLS exhibited enrichment of IFN-{gamma}, TNF-/NF-{kappa}B, p53, and metabolic pathways. Multiplex immunofluorescence further identified co-localization of exhausted immune cell populations within the core and periphery of peri-tumoral TLS. These findings indicate that a pro-tumorigenic microenvironment associated with disease progression in bladder cancer exists within peri-tumoral TLS and potentially a factor underlying contrasting therapeutic associations potentially driven by live microbial versus targeted immunomodulatory therapy in NMIBC and MIBC.

Activation of the STING pathway can induce potent antitumor immunity, but effective delivery of STING agonists to the tumor while limiting systemic exposure remains challenging. We previously developed MUSIC, an ultrasound-guided platform that uses microbubbles (MBs) to deliver the STING agonist 2'3'-cGAMP and locally activate antitumor immunity. However, the vascular confinement of MBs and the need for intratumoral administration limit the potential for systemic tumor targeting. To overcome these limitations, we developed SONATA (Systemic Oncotherapy using Nanobubbles for Acoustically-guided Tumor Activation), which employs nanobubbles (NBs) that are approximately 10-fold smaller than conventional MBs, enabling systemic administration and tumor extravasation. Following NB accumulation within tumors, ultrasound exposure triggers localized cGAMP release, facilitating delivery to targeted CD11b+ antigen-presenting cells (APCs) and STING activation with spatial and temporal control. NBs are composed of the same components as MBs, including phospholipid shells and a perfluorobutane core and are functionalized with anti-CD11b antibodies to target CD11b+ APCs and spermine-modified dextran to stably load cGAMP through nanocomplex formation. Upon ultrasound activation, SONATA induced phosphorylation of STING, TBK1, and IRF3 and increased IFN-{beta} production in bone marrow-derived macrophages. In an orthotopic breast cancer model, intravenously administered SONATA combined with tumor-localized ultrasound significantly inhibited tumor growth compared with controls. Furthermore, SONATA synergized with immune checkpoint blockade prolonged the median survival of tumor-bearing mice. Collectively, these findings establish SONATA as a systemically administered immunotherapy platform that enables ultrasound-guided, spatially controlled STING activation.

BACKGROUND: Cerebral ischemia following subarachnoid hemorrhage (SAH) has traditionally been considered transient because functional alterations of the cerebral microcirculation are thought to be self-limiting. However, we identified a previously unrecognized vasculopathy, perivascular fibrosis of the cerebral microcirculation (PFCM), characterized by excessive type I collagen deposition after SAH. This study investigated the mechanisms underlying PFCM and its subsequent effects on cerebral hemodynamics. METHODS: In vivo SAH was modeled in mice by autologous blood injection, whereas oxygenated hemoglobin (OxyHb) exposure was used to mimic SAH in vitro. Pericyte-deficient mice (Pdgfr{beta}+/-) and pericyte-specific vestigial-like family member 3 (VGLL3) conditional knockout mice (Vgll3{Delta}PC) were generated. Pericyte contractility was measured by nanoindentation and traction force microscopy. Molecular mechanisms were examined using Western blotting, immunofluorescence, CUT&Tag, RNA-seq, transmission electron microscopy, and molecular docking. PFCM, impaired dilation of the cerebral microcirculation, and cerebral autoregulation were assessed by two-photon imaging, transcranial Doppler with continuous blood pressure monitoring, super-resolution ultrasound imaging, and photoacoustic imaging. RESULTS: After SAH, mice developed long-term cerebral autoregulation dysfunction marked by impaired dilation of the cerebral microcirculation, with the abnormality being most evident within the relatively lower blood pressure range. The marked reduction in PFCM in Pdgfr{beta}+/- mice indicated that pericytes were the principal cellular contributors. Mechanistically, OxyHb-induced cytoskeletal remodeling in vitro increased pericyte contractility and promoted nuclear translocation of SAH-upregulated VGLL3. This was followed by increased genomic occupancy, Col1a1 transcriptional activation, and type I collagen deposition. Pericyte-specific VGLL3 knockout abolished PFCM and, consequently, significantly alleviated long-term cerebral autoregulation dysfunction. CONCLUSIONS: Our findings identify PFCM mediated by pericytic VGLL3 as a novel vasculopathy leading to long-term cerebral autoregulation dysfunction after SAH.

Despite significant advancements in microsurgical techniques in recent years, the treatment and prognosis of craniopharyngiomas remain unsatisfactory. As a central nervous system tumor located adjacent to important brain structures such as the hypothalamus-pituitary axis and accompanied by a highly inflammatory microenvironment, the tumor heterogeneity and tumor microenvironment characteristics of papillary craniopharyngiomas (PCPs) remain unclear. In this study, we integrated multimodal single-cell and spatial profiling from PCP tissue and peripheral blood mononuclear cells (PBMCs) to elucidate the tumor heterogeneity and microenvironment characteristics of PCP. Our single-cell and spatial analyses defined four specific tumor cell states in PCP, representing specific transcriptional regulatory programs and spatial heterogeneity characteristics during tumor progression. By constructing a spatial niche composed of tumor, immune, and stromal cells, we analyzed the cellular and spatial ecosystem of PCP at multiple levels to further assess the communication relationships between different tumor cell states and microenvironment cells. This study established a multidimensional molecular atlas of PCP from the perspectives of cell state, spatial structure, and microenvironment interactions, providing a foundation for understanding its biological behavior and exploring new intervention strategies.

BackgroundDedifferentiated liposarcoma (DDLPS) is characterized by abundant immune cell infiltration yet derives limited benefit from immune checkpoint blockade and stimulator of interferon genes (STING) agonist-based strategies, suggesting tumor-mediated suppression of antitumor immunity. Tumor-associated macrophages are the most abundant immune populations in DDLPS, but the factors regulating their function remain incompletely understood. MethodsExtracellular vesicles (EVs) were isolated from two DDLPS cell lines and serum from 16 DDLPS patients and 13 healthy donors. EVs impact on cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) -induced macrophage activation was assessed by cytokine secretion, surface markers, functional assays and macrophage-T-cell coculture. Proteomics was performed in EV-treated and EV-untreated macrophages from three donors. Pathway and protein interaction analyses were integrated with The Cancer Genome Atlas (TCGA) DDLPS transcriptomic and survival data. ResultsWe show that EVs released by DDLPS cells suppress macrophage responsiveness to classic STING agonist cGAMP. EVs derived from DDLPS attenuated cGAMP-induced expression of type I interferon-associated cytokines and chemokines, reduced IFN-{beta} secretion, and impaired phosphorylation of STING, TBK1 and IRF3. Functionally, DDLPS EV exposure shifted macrophages toward an immunoregulatory phenotype, restrained phagocytic activity, and attenuated macrophage-dependent T-cell proliferation while promoting T-cell exhaustion. Proteomic profiling revealed extensive macrophage reprogramming characterized by suppression of STING-associated signaling, antigen processing and presentation associated pathways and proteins targeted by miR-16-5p. Consistent with these findings, STING expression was associated with prolonged overall survival in DDLPS, while reduced expression of miR-16-5p target proteins was associated with attenuated STING pathway activity and immunostimulatory macrophage signatures. ConclusionsThese findings identify EV-mediated suppression of macrophage STING signaling as a mechanism of immune dysfunction in DDLPS and provide a framework for understanding immune resistance in this disease.

BackgroundOwing to their potent immunomodulatory properties, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (EVs) have emerged as promising neuroprotective treatments for ischemic stroke. Preclinical studies using MSC-EVs have mainly been performed in young, otherwise healthy rodents. Stroke patients frequently carry vascular risk factors and comorbidities. We herein investigated whether MSC-EVs retain neuroprotective activity in hyperlipidemic mice on cholesterol-rich Western diet. MethodsMale C57BL/6J mice were exposed to regular normal diet or Western diet for 6 weeks. At the age of 9-10 weeks, mice were exposed to transient intraluminal middle cerebral artery occlusion (MCAO). Vehicle or MSC-EVs (2x106 or 6x106 cell equivalents) were intravenously administered immediately after reperfusion, and vehicle or rosuvastatin (5 mg/kg/day) were intraperitoneally applied starting immediately after or seven days before MCAO. Neurological deficits, ischemic injury, and immune responses were evaluated up to 72 hours post-ischemia. To investigate the hyperlipidemia-associated immune dysregulation, mice received DNase-I before or immediately after MCAO. In defined subgroups, monocytes/ macrophages or neutrophils were additionally depleted by clodronate liposomes or anti-Ly6G antibodies, respectively. ResultsIn contrast to normolipidemic control mice, MSC-EVs failed to induce post-ischemic neuroprotection in hyperlipidemic mice. Neither MSC-EV dose escalation nor rosuvastatin co-treatment restored the therapeutic efficacy of MSC-EVs. Hyperlipidemia induced systemic innate immune dysregulation characterized by reduced monocyte/ macrophage activation, increased neutrophil activation, and elevated circulating cell-free DNA. DNase-I treatment before, but not after MCAO reversed these immune abnormalities and restored neuroprotection by MSC-EVs, decreasing neurological deficits, infarct volume and brain edema. Depletion of either monocytes/ macrophages or neutrophils abolished the neuroprotective effects of MSC-EVs in DNase-I-pretreated hyperlipidemic mice. ConclusionsImmune dysregulation abolishes MSC-EV-induced neuroprotection after ischemic stroke in hyperlipidemic mice. DNase-I priming restores MSC-EV responsiveness through mechanisms critically involving monocyte/ macrophage and neutrophil rebalancing. Our data highlight the host immune status as determinant of EV therapeutic efficacy.

Radiation therapy is a cornerstone of breast cancer treatment and reduces recurrence overall. However, patients with triple negative breast cancer (TNBC) continue to experience recurrence at higher rates than patients with other subtypes, especially when immunocompromised. While CD8 T-cells are known to mitigate recurrence, the role of CD4+ T-cell subsets in shaping the irradiated microenvironment remains unclear. We show that irradiated mammary tissue from mice accumulates CD4+ T-cells and exhibits a TGF{beta}-enriched cytokine milieu coincident with macrophage infiltration. We demonstrate that Th2-polarized CD4+ T-cells promote invasion of TNBC cells and macrophages through secretion of TGF{beta}. Neutralization of TGF{beta} significantly reduces this invasive phenotype. Mechanistically, Th2-conditioned media induces Tgfb1 expression in both TNBC cells and macrophages, establishing a TGF{beta}-dependent feed-forward amplification loop. In TNBC cells, Th2-derived TGF{beta} activates non-canonical signaling characterized by increased p38 MAPK and NF-{kappa}B phosphorylation, linking cytokine exposure to pro-invasive behavior. Together, these findings identify Th2-derived TGF{beta} as a driver of pro-invasive tumor reprogramming and suggest that interruption of Th2-TGF{beta} signaling may prevent recurrence following therapy.

Herein, we assess the potential of low-dose stereotactic radiosurgery (SRS) to modulate neuronal network states without apparent damage to cellular integrity. Using a small animal radiation research platform (SARRP), a 1 mm3 focal target in the mouse visual cortex was irradiated with doses of 5, 20, and 40 Gy. One-month later a significant dose-dependent increase in excitatory synapse numbers was observed, notably limited to the treated visual cortex and not the adjacent somatosensory cortex. Six months post-irradiation, cortical neuronal microcircuit activity was monitored in awake mice using high sensitivity two-photon calcium imaging. A single 5 Gy dose resulted in a significant microcircuit-wide increase of spontaneous neuronal activity, consistent with a lasting shift in the functional architecture of the irradiated nodal network. At higher SRS doses (40 Gy) this neuromodulatory window appears to close. In aggregate, these data suggest that low-dose radiation could, in some circumstances, be exploited by selected high precision SRS technologies to durably modulate neuronal circuit disorders. Some, or even all the clinical benefits reported in the companion article by Zhao et al. are likely attributable to the biological properties we sought to characterize in our research. One Sentence SummaryLow-dose stereotactic radiosurgery effectively and durably modulates neuronal excitability via synaptic re-organization and could open new clinical possibilities for neuromodulation.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor microenvironment (TME) with pancreatic myofibroblasts (PMF) and macrophages being two prominent cell populations essentially impairing tumor responses to (immuno)therapies. L1 cell adhesion molecule (L1CAM) is upregulated in PDAC cells in primary and metastatic tissues and associated with tumor progression and therapy resistance. Using L1CAM as tumor-associated antigen, two bispecific antibodies (bsAB) targeting L1CAM and CD3 were developed in the IgG-(L)-ScFv format and their anti-tumorigenic activity was investigated in different preclinical PDAC models. In 2D models, both L1-bsAB exerted L1CAM-specific anti-PDAC cell activity when co-cultured with activated CD8+ T cells. Strong anti-PDAC cell effects along with elevated release of T cell effector molecules were also observed upon co-culture with peripheral blood mononuclear cells (PMBC) from healthy donors and PDAC patients. Of note, both L1-bsAB were also effective in 3D PDAC cell spheroids and neither impaired by PMF nor macrophages. Finally, application of L1-bsAB on organotypic tissue slice cultures from PDAC tissues comprising the entire complex TME also induced PDAC cell apoptosis and release of T cell effector molecules. Overall, our results highlight relevant anti-PDAC cell activity of L1-bsAB in immunosuppressive contexts supporting their potential as immunotherapeutic strategy for PDAC.

Mitochondrial plasticity, characterized by the dynamic balance between glycolysis and oxidative phos-phorylation in response to genetic and microenvironmental changes, is a hallmark of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations had shown that the lysosomal sphingolipid-metabolizing enzyme {beta}-galactosylceramidase (GALC) rewires the lipid profile of mouse melanoma cells, exerting pro-oncogenic functions, gene silencing leading to a decreased oncogenic activity in murine and human melanoma cells. Here, we have focused on the mitochondrial sphingolipid composition and energetic metabolism in GALC knockout (KO) A2058 human melanoma cells. Targeted analysis of the mitochondrial sphingolipid profile, transcriptomic data, and mitochondrial structural and functional studies indicate that GALC loss drives a sphingolipid-mediated reprogramming of mitochondrial metabolism in absence of major structural alterations, characterized by bioenergetic insufficiency possibly due to ceramide- and sphingomyelin-driven impairment of respiratory chain function. Overall, these data indicate that GALC KO leads to a sphin-golipid-driven mitochondrial metabolic suppression and may provide novel information for the development of efficacious approaches in mitochondrial targeting melanoma therapies.

Abstract Background: Cytolytic activity (CYT), a widely used transcriptomic surrogate of anti-tumor immune cytotoxicity derived from GZMA (granzyme A) and PRF1 (Perforin 1) expression, is associated with clinical outcomes across cancers. MicroRNAs (miRNAs) are key post-transcriptional regulators of tumor immunity, yet their pan-cancer roles in modulating cytolytic activity remain incompletely understood. Objective: This study aimed to identify conserved miRNA regulators of tumor cytolytic activity and their downstream gene-mediated networks across diverse cancer types, while evaluating their clinical and therapeutic relevance. Methods: Matched miRNA and mRNA expression profiles from 9,288 primary tumors across 31 TCGA cancer types were analyzed. A multi-stage framework was applied: per-cancer Spearman correlations (|{rho}| >= 0.30, FDR < 0.05) identified recurrent CYT-associated miRNAs (at least 3 cancer types); these were integrated with TargetScan-predicted targets and subjected to pan-cancer and cross-cancer triple filtering (miRNA-gene and gene-CYT associations). All associations underwent tumor purity adjustment using Consensus Purity Estimate (CPE), with LUMP (Leukocytes Unmethylation for Purity) as sensitivity analysis. Candidates were further prioritized by random forest modeling with bootstrap stability, cancer-type-adjusted Cox regression, mediation analysis, immune cell deconvolution, k-means molecular subtyping, pathway enrichment, and DGIdb-based drug-target prioritization. Results: The analysis converged on 38 high-confidence miRNA-gene-CYT regulatory triplets involving 9 conserved miRNAs and 31 target genes after stringent purity adjustment and multi-layer validation. All nine miRNAs exhibited complete bootstrap stability. Mediation analysis confirmed significant gene-level mediation in 37 of 38 triplets (FDR < 0.01), with mediated proportions up to 94%. The final miRNA signature defined two distinct pan-cancer immune subtypes (immune-hot vs. immune-cold) with significantly different cytolytic activity and overall survival (OS) (HR = 0.754, FDR = 1.12 x 10^-4). The network was enriched for T-cell activation and lymphocyte differentiation pathways and highlighted multiple druggable targets, including CTLA4 and CD274 (PD-L1), nominating 124 candidate compounds. Conclusions: In conclusion, this tumor purity-adjusted pan-cancer study defines a compact, reproducible, and clinically relevant miRNA network that regulates cytolytic activity across diverse malignancies. By linking miRNA biology to immune subtyping and actionable therapeutic targets, the present work provides a valuable foundation for advancing precision immuno-oncology.

Glioblastoma remains highly lethal, with median survival of ~15 months. Resistance to temozolomide is ubiquitous, yet its mechanisms are incompletely understood. Here, we identify the CCL20-CCR6 chemokine axis as a stress-responsive survival pathway limiting therapeutic efficacy. Targeting CCL20-CCR6 in combination with temozolomide and cannabidiol was evaluated using clinical datasets, GBM cell lines, tumor organoids, and a syngeneic CT-2A mouse model integrating proteomic and lipidomic profiling. Low CCL20 expression was associated with improved survival, supporting its prognostic relevance. Across models, TMZ alone or with CBD induced CCL20 expression while exerting limited antitumor activity. Targeted disruption of CCL20-CCR6 signaling using dendrimer-delivered shRNA enhanced therapeutic response in murine models and GBM organoids. Multi-omic analyses revealed that CCL20 inhibition reprograms the tumor microenvironment and induces mitochondrial dysfunction, resulting in elevated reactive oxygen species (ROS) and tumor cell death. This effect was accompanied by accumulation of 17-hydroxydocosahexaenoic acid and activation of oxidative stress-associated cytotoxic pathways. Functional assays confirmed that CCL20 blockade selectively amplifies mitochondrial ROS beyond levels induced by TMZ alone potentiating TMZ efficacy by promoting mitochondrial oxidative stress. Targeting this axis represents a promising strategy to overcome chemoresistance and positions CCL20 as both a prognostic biomarker and a therapeutic vulnerability in GBM.