Theranostics

Androgen receptor (AR) antagonists, such as enzalutamide, suppress prostate cancer (PCa) cells to achieve temporary therapeutic effects. In addition to tumor cell-autonomous suppressive function, AR antagonists can also potentially exert anti-tumor effects via mitigating cytotoxic T cells exhaustion. However, strategies for effectively harnessing enzalutamides immunotherapeutic effects remain elusive. In studying a recently described glutamine antagonist prodrug (DRP-104) in PCa models, we found that despite the initial response, tumors ultimately became resistant. Intriguingly, compared to the untreated (DRP-104 treatment-naive) tumors, the resistant tumors became highly susceptible to enzalutamide in vivo. Additionally, treating tumors with DRP-104 and enzalutamide simultaneously also yielded superior therapeutic efficacy. We demonstrated that DRP-104 therapy promoted the infiltration of CD8+ T cells as well as regulatory T cells (Treg) in responsive tumors, and the tumor-infiltrating Treg were mostly depleted upon enzalutamide treatments. Mechanistically, we showed that Treg differentiation from mouse CD4+ T cells was attenuated by enzalutamide. We further demonstrated that Treg induction was accompanied by the interaction between AR and aryl hydrocarbon receptor (AhR), the nuclear receptor indispensable for Treg differentiation, in the nuclei of CD4+ T cells, and this interaction was diminished by enzalutamide. In further support of AR signaling in Treg biogenesis, analysis of available gene expression datasets found that AR expression was elevated in Treg when compared to CD4+ T cells in human peripheral blood mononuclear cells (PBMCs). In addition, it was positively correlated with Treg module scores in several human cancer types. Finally, using an anti-GPC3 (Glypican 3) vaccination model, we demonstrated that CD4+ T cells subjected to Treg induction in the presence of enzalutamide were less effective in protecting GPC3-expressing tumor cells from CD8+ T cells cytotoxic killing. Collectively, these results suggest that AR promotes Treg s differentiation and/or immunosuppressive functions, and nominate enzalutamide as a Treg-mitigating agent for potentiating immunotherapies. Our results also demonstrate that an otherwise unintended, Treg-promoting property of DRP-104 can be leveraged to unleash the immune-regulatory function of enzalutamide for the treatment of PCa.

Chimeric antigen receptor (CAR) T cell therapy has limited efficacy against solid tumors such as prostate cancer due to the immunosuppressive tumor microenvironment (TME). Combining CAR T cells with existing therapies that remodel the TME and promote endogenous immune responses, such as radiation therapy and chemotherapies, may strengthen antitumor responses. Here, we assessed the potency of combining focal radiotherapy (RT), cyclophosphamide (Cy) preconditioning, and prostate stem cell antigen (PSCA)-CAR T cells against syngeneic prostate cancer models. Focal RT alone increased T cell and dendritic cell infiltration and activation in the irradiated tumor. Furthermore, the combination of all three therapies was critical for enhanced antitumor responses and survival across multiple subcutaneous, bone-metastatic, and multifocal disease models. This combination, in the irradiated TME and tumor-draining lymph nodes (tdLN), led to greater antigen presentation by myeloid cells and endogenous T cell activation and cytotoxicity. Our study demonstrates the potency of combining focal RT with PSCA-CAR T cells, significantly improving therapeutic responses in the irradiated tumor and contributing to a more robust systemic immune response against metastatic burden in prostate cancer.

Background: Cutaneous melanoma is the most aggressive malignant skin tumor, and metastasis represents the primary cause of patient mortality. Bisphenol S (BPS) has an unclear influence on melanoma metastasis and its underlying molecular mechanisms. Methods: Potential BPS targets were predicted using the SEA, SwissTargetPrediction, and SuperPred databases. Based on TCGA-SKCM transcriptomic data, differential expression analysis was performed, and Weighted Gene Co-expression Network Analysis (WGCNA) was employed to construct a gene co-expression network. Candidate genes were obtained by integrating BPS-related targets, differentially expressed genes (DEGs), module genes, and univariate Cox regression genes, followed by Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network construction. Least Absolute Shrinkage and Selection Operator (LASSO)-Cox regression was applied to screen core prognostic genes and construct a risk prediction model. Further analyses included network construction, molecular docking, and 100 ns molecular dynamics (MD) simulation. Results: Integration of BPS-related targets, DEGs, WGCNA module genes, and Cox regression results yielded 13 candidate genes enriched in kinase activity regulation and melanoma-related pathways. LASSO-Cox regression ultimately identified three core prognostic genes--ABCB1, PIM2, and TSHR--all significantly upregulated in metastatic tissues, with area under the curve (AUC) values of approximately 0.7. High-expression patients exhibited significantly better overall survival than low-expression patients (P < 0.05). A nomogram incorporating the three genes and clinical parameters demonstrated good calibration performance. Within the ceRNA network, MALAT1 and hsa-miR-155-5p were identified as key regulatory molecules, and 37 potential transcription factors were predicted, including CEBPA, JUN, and STAT3. Molecular docking revealed strong binding affinities of BPS toward ABCB1 , PIM2, and TSHR, and MD simulations confirmed the structural stability of all three complexes. Conclusion: ABCB1, PIM2, and TSHR are the core target genes through which BPS influences melanoma metastasis via multidrug resistance, kinase signaling, and receptor-mediated signal transduction. The prognostic model based on these three genes demonstrates good clinical applicability, and the ceRNA and transcription factor regulatory networks provide a systematic molecular basis for understanding the association between BPS exposure and melanoma metastasis.

177Lu-PSMA-617 (PluvictoTM, Lu-177 RLT) is an FDA-approved targeted radioligand therapy (RLT) for metastatic castration-resistant prostate cancer (mCRPC), but its durability of response to this singular approach poses a challenge to the field. Chimeric antigen receptor (CAR) T cell therapy has revolutionized clinical practice for hematological malignancies, but its clinical development for solid tumors, including mCRPC, has been encumbered by antigen heterogeneity and the immunosuppressive tumor microenvironment (TME). Here, we evaluate the therapeutic combination of Lu-177 RLT and PSCA-CAR T cells to overcome these barriers. In human xenograft and mouse syngeneic prostate cancer models with homogeneous or heterogeneous antigen expression, the sequential administration of Lu-177 RLT, cyclophosphamide (Cy), and PSCA-CAR T cells improves tumor control and prolongs survival compared to monotherapies. Mechanistically, Lu-177 RLT alone or with Cy remodels the TME by promoting pro-inflammatory myeloid responses and activating endogenous T cells, while enhancing CAR T cell activation and effector function. We additionally evaluated 225Ac-PSMA-617 RLT as an emerging approach in combination with CAR T cells and observed anti-tumor responses, supporting its potential as an alternative RLT partner. These findings support RLT as an immune priming strategy to enhance CAR T cell therapy and provide a rationale for clinical translation of this combination in mCRPC. One Sentence SummaryCombining 177Lu-PSMA-617 radioligand therapy with PSCA-CAR T cells improves tumor control and survival in prostate cancer models by overcoming the antigen heterogeneity and reshaping the immunosuppressive tumor microenvironment.

Glioblastoma (GBM) remains one of the most lethal primary brain tumors, in part because the blood-brain barrier (BBB), restricts delivery of most systemically administered chemotherapeutics. Although focused ultrasound (fUS) can transiently increase BBB permeability, therapeutic efficacy remains limited by reliance on systemic drug exposure and heterogeneous intratumoral distribution. Here, we report a pressure-gated ultrasound-triggered drug delivery strategy that enables localized intravascular release of chemotherapy at the site of sonication. Freebase doxorubicin and afatinib were encapsulated within ultrasound-sensitive mPEG-PDLLA/PFOB microdroplets and administered systemically to N-TVA::Ink4a/Arflox/lox;Ptenlox/lox mice bearing genetically engineered glioblastomas. Animals received repeated transcranial focused ultrasound over a 30-day treatment period. Ultrasound-triggered release of the dual-drug formulation significantly extended survival compared with untreated controls, with median survival increased by over two weeks - approximately a 30% improvement. Furthermore, this survival improvement was reflected in histological analysis, showing decreased tumor burden and severity. These improvements were not found in any control groups, demonstrating that spatially and temporally controlled intravascular drug release can substantially improve therapeutic efficacy in an aggressive immunocompetent glioblastoma model. These findings support pressure-gated ultrasound-triggered chemotherapy as a promising activation-based strategy for overcoming BBB-associated delivery limitations and improving outcomes in malignant brain tumors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/735435v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1767043org.highwire.dtl.DTLVardef@c46048org.highwire.dtl.DTLVardef@8d3b44org.highwire.dtl.DTLVardef@2df0b8_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPressure-gated focused ultrasound enables localized release of doxorubicin and afatinib in glioblastoma. C_LIO_LIUltrasound-triggered chemotherapy significantly extends survival in a genetically engineered immunocompetent GBM model. C_LIO_LILocal activation outperforms systemic administration of identical drug combinations. C_LIO_LIThis strategy shifts focused ultrasound therapy from general BBB opening to spatially controlled drug activation. C_LI

Cytotoxic T lymphocytes (CTLs) play a central role in antitumor immunity; however, metabolic reprogramming within the tumor microenvironment often compromises their effector function, making metabolic targeting crucial for the improvement of T cell function. Folate-dependent purine synthesis, a core pathway sustaining the nucleotide pool, is highly activated in tumors, yet its role in regulating tumor immune sensitivity remains unclear. Here, by establishing a co-culture system of melanoma cells and human T Cell Receptor (TCR)-engineered T cells, we systematically evaluated the effects of folate-dependent purine synthesis inhibitors on tumor cell response to CD8+ T cell cytotoxicity. We found that inhibition of key enzymes such as methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and glycinamide ribonucleotide transformylase (GART) markedly enhanced tumor cell sensitivity to T cell killing, an effect also observed with exogenous nucleoside supplementation. Mechanistically, inhibition of folate-dependent purine synthesis suppresses glycolysis by downregulating critical glycolytic enzymes, thereby reducing lactate production. Reduction in lactate further weakens lactylation and stability of the immune checkpoint protein PD-L1. In parallel, impaired purine synthesis disrupts uridine metabolism, blocks ribose salvage, and distally influences glycolysis. Collectively, our study identified the folate-dependent purine synthesis-glycolysis axis as key regulator of tumor immune response and highlights metabolic targeting as a promising strategy to improve cancer immunotherapy.

The epidermal growth factor receptor (EGFR) expression is often dysregulated in head and neck squamous cell carcinoma (HNSCC), driving cancer cell proliferation, invasion, and metastasis through diverse pathways, thereby contributing to aggressive chemo- and radio-therapy resistance. A GPI-anchored protein, CD109 is upregulated in multiple cancers, including HNSCC. While membrane-anchored CD109 (mCD109) is pro-tumorigenic in SCC via EGFR/STAT3 activation, the role of protease-cleaved soluble CD109 (sCD109) is poorly understood. Our groundbreaking findings demonstrate that sCD109 antagonizes EGFR signaling by directly binding to the EGFR extracellular domain, preventing mCD109-EGFR stabilizing interactions on the cell surface, followed by inhibition of EGFR phosphorylation at Y1068 and downstream signaling cascades (AKT, MAPK, and STAT3) consequently suppressing cancer cell migration, invasion, 3D tumor spheroid formation and angiogenic tube formation. In addition, we found that sCD109 regulates EGFR fates by inhibiting nuclear localization of phosphorylated EGFR and promoting EGFR degradation. Additionally, sCD109 significantly reduces EGF-induced expression of cancer stem cell markers (CD44 and CD133) and embryonic stem cell markers (Nanog and Sox2), suggesting a suppressive role in cancer stemness. Taken together, these results underscore the opposing roles of mCD109 and sCD109: with sCD109 acting as an antagonist by inhibiting mCD109/EGFR-driven oncogenic signaling and phenotypes. Our current findings reveal a complex interplay among mCD109, sCD109, and EGFR, identifying a mechanism for targeting EGFRs degradation in HNSCC, and lay the groundwork for future research on investigating sCD109s modulatory role in preclinical models of HNSCC.

Pancreatic cancer remains a highly lethal malignancy with limited therapeutic options. Chimeric antigen receptor (CAR) therapy has revolutionized the treatment of hematological cancers but still faces major limitations in solid tumors, particularly due to the scarcity of tumor-specific targets. Cutaneous lymphocyte antigen (CLA) recently emerged as a promising PDAC target due to its high tumor expression and limited presence in healthy tissues. However, previously reported CLA-directed CAR constructs lacked antitumor functionality. Here, we investigated multiple strategies to generate functional CLA-targeting CAR approaches. We first hypothesized that impaired activity resulted from fratricide caused by CLA expression on activated T cells. CLA knockout was successfully achieved through deletion of fucosyltransferase-7, but not by knockout of the major CLA carrier backbones CD162, CD44 or CD43, suggesting additional CLA carriers or compensatory regulation. As CLA knockout alone did not restore CAR-mediated killing, we explored whether insufficient binding affinity limited CAR activity. Affinity maturation was performed in silico and in vitro using yeast surface display, identifying 39 candidate mutations, although none restored cytotoxicity. We finally switched to an AdCAR strategy using anti-biotin CAR T cells combined with biotinylated anti-CLA scFv-Fc adapters. This approach enabled efficient, concentration-dependent cytotoxicity with both CLA-targeting binders. Additionally, we identified a dynamic, cell density-dependent regulation of CLA expression. Finally, glycan profiling of CLA binders further revealed broader-than-expected glycan interactions, suggesting a potentially wider definition of the CLA family. Overall, our findings establish CLA as a functional PDAC immunotherapy target while revealing unexpected complexity in its regulation and molecular presentation.

Ischemic stroke remains a leading cause of death and disability, and current reperfusion therapies do not address the secondary neuroinflammatory response following blood-brain barrier (BBB) disruption. Purinergic signaling critically regulates this process: extracellular ATP promotes inflammation, whereas its enzymatic conversion into adenosine exerts tissue-protective effects. Notably, the ectonucleotidase CD73 (NT5E), which catalyzes AMP-to-adenosine conversion, is highly expressed by human but not murine brain endothelial cells (BECs). Here, we investigated the role of endothelial CD73 in ischemic stroke using an AAV vector engineered for selective transduction of murine BECs to induce BBB-specific CD73 expression. Endothelial CD73 enhanced extracellular ATP degradation toward adenosine generation and established a purine metabolism profile resembling that of human BECs. In the transient middle cerebral artery occlusion (tMCAO) mouse model, BBB-targeted CD73 expression reduced infarct volume by 40% and prevented early mortality within 48 h after reperfusion. Transcriptomic and flow cytometric analyses revealed altered leukocyte responses, including increased recruitment of monocytes/macrophages whose gene expression signatures were consistent with inflammation-resolving programs. These findings identify endothelial CD73 as an important regulator of post-ischemic neuroinflammation and highlight species-specific differences in BBB purine metabolism with implications for translational stroke research.

Despite continuous progress in diagnosis and therapy, breast carcinoma (BC) remains a major health problem. Triple-negative (Estrogen Receptor-/Progesterone Receptor-/HER2-) breast cancer (TNBC) is the most aggressive subtype due to its high metastatic potential and resistance to chemotherapy. The Y-box binding protein 1 (YB-1) transcription factor, a protein present in both cytoplasm and nucleus, is a driver of TNBC malignancy as it stimulates its cancer stem cell phenotype and disrupts cell cycle progression. Here, we hypothesized that YB-1-containing sEVs deliver YB-1 to the nuclear compartment of recipient cancer cells and play a major role in the activation of the metastatic process. We found a selective enrichment of YB-1 in sEVs from MDA and 4T1 cells, with [~]65% and 50% of all sEVs positive for YB-1 by d-STORM. Administration of sEVs from wild-type MDA and 4T1 to their YB-1 knockout counterparts resulted in nuclear translocation of sEV-associated YB-1 and increased tumorsphere formation. Pharmacological blockade of the nuclear transport machinery based on the inhibition of the formation of the "VOR" complex (VAP-A-ORP3-Rab7) by PRR851 impaired both nuclear translocation and the YB-1-induced increase in tumorsphere formation. YB-1 phosphorylation at S102 was required for nuclear localization. In fact, loss of YB-1 phosphorylation inhibited tumorsphere growth and stemness of cancer cells and YB-1-positive sEVs restored the oncogenic behavior of cancer cells expressing phospho-mutant YB-1. Moreover, PRR851 inhibited the nuclear translocation of the phosphorylated form of YB-1 and the oncogenic behavior of the TNBC cells. These data support the conclusion that the nuclear translocation of sEV-associated phosphorylated YB-1 is an important factor in the malignant behavior of TNBC and a potential therapeutic target.

In vivo infrared thermography is limited by the inherently poor spatial resolution at long wavelengths, low contrast, and the lack of biocompatible contrast agents. Here, we present 3-5 m mid-wave infrared (MWIR) thermography enhanced by an artificial intelligence (AI) network and cold phosphate-buffered saline (PBS) as a thermal contrast agent for noninvasive in vivo imaging with high contrast and resolution. MWIR imaging enabled high thermal sensitivity with microscale spatial resolution, strong relative thermal contrast, and facilitated visualization of the subcutaneous vasculature in the human arm, hand, ankle, the femoral artery and vein in rats, and the femoral vessels in mice, with image contrast further enhanced by AI networks. In a 4T1 tumor-bearing mouse model, AI-enhanced MWIR resolved early-stage tumors of ~2.3 mm and metastases as small as ~1.7 mm. Using cold PBS as a MWIR thermal contrast agent, we achieved precise tumor boundary visualization and real-time imaging-guided tumor resection. AI-enhanced MWIR offers a promising solution for early diagnosis and improved surgical precision.

Neuroimmune crosstalk is increasingly recognized as a key regulator of tumor progression and therapeutic response, yet its role in central nervous system (CNS) tumors remains poorly understood. Here, we investigate tyrosine hydroxylase (TH)-mediated neuronal signaling in glioblastoma (GBM) and its impact on antitumor immunity and response to oncolytic virotherapy (OV). We show that TH cells are widely distributed within the GBM microenvironment, including neurons, astrocytes, and immune cells, and are enriched at the tumor margin. In addition, TH cells are present in the tumor-draining lymph nodes (TDLNs) of GBM, where they localize near lymphatic vessels and are associated with lymphangiogenesis. Notably, a subset of CD3TH T cells is detected within lymphatic structures of TDLNs, suggesting immune-intrinsic catecholamine signaling. Single-cell RNA sequencing reveals that noradrenergic signaling, particularly via {beta}2-adrenergic receptors (ADRB2), predominates in tumor-infiltrating myeloid cells and is dynamically regulated by therapy. Intratumoral administration of oncolytic herpes simplex virus (oHSV) upregulates ADRB2 expression in macrophages, whereas systemic chemo-immunotherapy induces distinct receptor modulation patterns in tumors and TDLNs. Functionally, pharmacologic {beta}-adrenergic blockade significantly enhances the efficacy of oHSV therapy in orthotopic GBM and subcutaneous melanoma models, resulting in reduced tumor growth, increased tumor cell death, and enhanced CD8 T cell infiltration. Similarly, direct inhibition of TH enzymatic activity suppresses tumor progression and further potentiates OV. Mechanistically, TH inhibition not only promotes tumor-infiltrated cytotoxic immune cells CD8, NK and {gamma}{delta} T cells, but also suppresses the activity of immunosuppressive myeloid cells, including transcriptional (Fos), and metabolism (Arg) modification in M2 macrophages and other immune cells. Collectively, these findings identify TH-mediated neuroimmune signaling as a critical regulator of tumor immunity in GBM and demonstrate that targeting catecholaminergic pathways or downstream neuroimmune crosstalk pathways can enhance the efficacy of OV. This study provides a rationale for integrating neural modulation into immunotherapeutic strategies for CNS malignancies.

Functional ultrasound (fUS) maps cerebral blood volume (CBV) but lacks molecular and neuronal specificity. By simultaneously integrating fUS with optical imaging, we show that fUS-derived CBV correlates with both optically measured hemoglobin and neuronal calcium activity in awake mice. We further derive hemodynamic response functions linking calcium activity to CBV during spontaneous and sensory-evoked activity. Application to a mouse glioblastoma model demonstrates utility for studying neurovascular dysfunction in complex neuropathologies.

Structural AbstractO_ST_ABSBackgroundC_ST_ABSTriple-negative breast cancer (TNBC) represents a major clinical challenge due to its aggressiveness, heterogeneity and limited availability of effective targeted therapy. We investigated whether LRP-1, a multifunctional cell-surface endocytic and signaling receptor, contributes to TNBC progression. MethodsUsing CRISPR-Cas9, LRP-1-deficient murine 4T1 and human HS578-T TNBC cells were used. Functional consequences were assessed through migration, invasion, and 3D spheroid assays, imaging of focal adhesions and actin organization, atomic force microscopy, and plasmin activity assays. Global molecular reprogramming was analyzed by label-free quantitative proteomics and secretomics. LRP-1-deficient or proficient 4T1 cells were implanted orthotopically in immunocompetent mice; tumor progression was monitored longitudinally while peritumoral collagen architecture and immune microenvironment composition were characterized by second harmonic generation imaging and immunohistochemistry. ResultsWe show that LRP-1 loss reduces TNBC aggressiveness, as reflected by decreased migration and invasive capacity, reduced spheroid evasion, and significant morphological changes in focal adhesion and actin structure. LRP-1-deficient cells became stiffer and showed lower LOXL-4 levels, while pericellular proteolytic activity remained unchanged, suggesting other proteases mechanism. Multi-omic analysis revealed alterations in extracellular matrix (ECM), epithelial-mesenchymal transition, and inflammatory pathways. In vivo, LRP-1-deficiency reduced tumor progression and peritumoral collagen deposition, while increasing CD8+ T and Natural Killer cell infiltration, together with a cytokine profiling compatible with a more immune-permissive microenvironment. ConclusionsLRP-1 act as a key contributor in TNBC progression through matrix remodeling, mechano-adaptation, and immune exclusion. Positioning it as a candidate biomarker for TNBC patients who are likely to benefit from stroma-targeting therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/732906v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1b595c2org.highwire.dtl.DTLVardef@7b208aorg.highwire.dtl.DTLVardef@1956e54org.highwire.dtl.DTLVardef@17e55d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pancreatic ductal adenocarcinoma (PDAC) is one of the most challenging cancers to treat due to the dismal survival rate, poor post-treatment outcome and profound resistance to a wide range of therapies. With mutant KRAS being a key driver, small molecule inhibitors targeting KRAS or pan-RAS (KRASi) have demonstrated exciting preclinical and early clinical anti-tumor efficacy, and the pan-RAS(ON) inhibitor daraxonrasib (RMC-6236) recently achieved Phase 3 clinically meaningful improvements in patient survival compared to chemotherapy. But resistance to RAS/KRAS inhibitor inevitably develops, which limits and compromises the treatment outcome. In this study, we investigated the combination of stereotactic body radiation therapy (SBRT) and KRAS inhibition (MRTX1133 and daraxonrasib) in the treatment of preclinical PDAC models. We found that this combination strategy synergistically suppresses PDAC cell growth in vitro and enhances tumor control while minimizing local recurrence in orthotopically implanted KPC (LSL-KrasG12D/+;Trp53R172H/+;Pdx1-Cre) murine PDAC tumors in vivo. As radiation therapy (RT) induces ferroptosis in multiple cancer types and mutant KRAS promotes various anti-ferroptotic mechanisms, we tested the role of ferroptosis in promoting tumor-control efficacy. Intriguingly, the addition of a ferroptosis inhibitor, liproxstatin-1, to the combination therapy significantly abrogated the in vivo synergism between SBRT and KRAS inhibition, suggesting that treatment-induced ferroptosis at least partially drives the synergistic efficacy of this combination strategy. Our study indicates that this SBRT-KRASi combination has the potential to overcome treatment resistance and improve outcomes in PDAC patients. These data directly support the design of a planned multi-center Phase 2 clinical trial with this combination strategy in locally advanced PDAC.

Brain metastases (BrM) are associated with poor prognosis. A better understanding of anti-tumor immune responses in the context of immune specialized microenvironment of the brain is required to develop improved therapeutic strategies for this disease. We demonstrate that the conventional dendritic cells type 1 (cDC1) gene signature positively correlates with a prolonged BrM-dependent survival in melanoma and breast cancer patients. Furthermore, intracranial anti-tumor immune responses in preclinical BrM models consistently rely on cDC1s for tumor growth control, BrM-dependent survival and maintenance of the intra-tumoral CD8+ T cell pool, in contrast to variable, cancer type-dependent cDC1 roles in extracranial tumors. This is underpinned by tumor site-specific cDC1 molecular profiles with distinct Toll like receptor repertoires, upregulation of co-stimulatory molecules and IL-12, and enhanced type-I-IFN signaling in intracranial cDC1s, with the latter driving increased cDC1 activation. cDC1s also promote the conversion of progenitor exhausted CD8+ T cells to transient effectors, which is further enhanced by immune checkpoint blockade therapy. These findings pinpoint cDC1s as a major cell population of interest in the development of future immunotherapies for BrM.

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

Background The ischemic penumbra, a metabolically compromised yet potentially salvageable region surrounding the ischemic core, is a prime target for acute stroke intervention. Yet an objective molecular definition of the penumbra, particularly during the earliest stages of ischemia, remains lacking. Methods and Results We applied principal component analysis (PCA) followed by k-means clustering to high-resolution mass spectrometry imaging data covering multiple metabolic pathways to identify a metabolically defined penumbra in a mouse model of hyperacute stroke (45 min middle cerebral artery occlusion, MCAO). Targeted spatial metabolomic profiling by matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) reveals a distinct penumbral metabolic profile, marked by relative preservation of high-energy phosphates, comparable lactate accumulation, and reduced succinate accumulation relative to the core. Spatial transcriptomics revealed selective induction of immediate-early genes, including Npas4, Fos and Junb, within the penumbra. Consistently, imaging mass cytometry shows enrichment of phospho-histone H3 (pHH3) within the penumbra, suggesting a chromatin-associated response potentially linked to immediate-early gene activation. Conclusion Together, these findings provide a multimodal molecular atlas of the hyperacute metabolically defined penumbra and reveal molecular features that facilitates its identification and inform future therapeutic strategies.

Accurate assessment of cerebral hemodynamics impairment traditionally relies on arterial metrics, yet often overlooks venous drainage and arteriovenous dynamics, thereby limiting the evaluation of ischemia-induced microvascular dysfunction. To address this limitation, we implemented a signal-averaging framework, combined with co-fluctuation analysis, to extract predominantly arterial and venous hemodynamic signals and construct a dynamic arteriovenous co-fluctuation index that quantifies frame-by-frame coordination between arterial inflow and venous outflow activity. This time-resolved index enables spatial characterization of large-scale cortical arteriovenous coordination beyond conventional static correlation-based analyses. Comparative analyses between healthy controls and acute ischemic stroke mice demonstrated that the arteriovenous co-fluctuation index sensitively detects disruption of vascular coordination, revealing a slower state transition that occurs alongside distinct temporal abnormalities and regional heterogeneity between ischemic core and penumbral regions. These findings underscore the utility of arteriovenous coordination as a sensitive indicator of microcirculatory dysfunction, offering a practical analytical tool for assessing stroke-induced microvascular impairment.

Chemotherapy resistance remains a critical barrier in treating osteosarcoma. Hypoxia-activated prodrugs (HAPs) target oxygen-deprived tumor regions that evade conventional chemotherapy. Here, we applied integrated spatial multimodal mass spectrometry imaging of metabolites (DESI-MSI, MALDI-MSI), targeted proteomics (IMC), and metallomics (LA-ICP-MSI) to naive and newly developed doxorubicin-resistant osteosarcoma spheroids treated with a novel HAP tirapazamine analogue, TPZ-A-X, and doxorubicin. Combination treatment selectively downregulated GLUT1 and suppressed pro-survival pAkt in doxorubicin-resistant spheroids whilst inducing comparable DNA damage ({gamma}H2AX) across both phenotypes. Metabolomics imaging identified ferroptosis pathway suppression in doxorubicin resistance, which combination treatment reversed, whilst simultaneously depleting glycolytic fuels. Integrative protein-metabolite correlation analysis uncovered functional couplings between glucose transport and CoA-dependent metabolism and spatially revealed anabolic signaling at spheroid peripheries. Combination treatment induced endogenous copper, zinc and magnesium depletion, independent of ATP/ADP collapse reflecting an adaptive survival remodeling of the metalloproteome. HAP/chemotherapy combinations exploit metabolic vulnerabilities via coordinated disruption of ferroptosis suppression, glycolytic dependence, and survival pathways underlying apoptotic resistance. These findings demonstrate a framework for informing mechanistic reasoning and combination strategy design in chemotherapy-resistant tumors.