Abstract Brown adipose tissue (BAT) is a mitochondria-rich thermogenic organ whose function depends on high oxidative capacity, yet how primary mitochondrial dysfunction remodels BAT identity and metabolism remains poorly defined. Using the Deletor mouse model of progressive mtDNA deletion disease, we identify a pseudohypoxia-iron-NAD + axis as a central organiser of BAT pathology. Deletor BAT underwent profound structural, transcriptional and metabolic remodelling, characterised by mitochondrial ultrastructural damage, loss of thermogenic identity, PHD3/HIF-associated pseudohypoxic signalling, iron dysregulation and NAD + /NADH redox imbalance. Indirect calorimetry confirmed that this molecular disease program translates to functional thermogenic failure under physiological demand. Whole-body indirect calorimetry showed reduced oxygen consumption during acute cold challenge and impaired RER-based fuel switching, consistent with impaired cold-adaptive oxidative metabolism. Metabolomic profiling revealed altered TCA cycle intermediates, glycolytic rewiring and selective amino acid accumulation. Pharmacological perturbation showed that the PHD inhibitor roxadustat worsened disease-associated features, whereas HIF-1α suppression with PX-478 attenuated the integrated stress response, indicating that pseudohypoxic signalling is maladaptive in this setting. Nicotinamide riboside broadly attenuated the disease metabolome and transcriptome, restoring NAD + /NADH balance, suppressing ISRmt, iron-stress and pseudohypoxic gene programs, and correcting selective carnitine and acylcarnitine abnormalities consistent with impaired fatty-acid handling. These findings define a therapeutically tractable pseudohypoxia-iron-NAD + axis as a core determinant of BAT dysfunction in mitochondrial disease.

Endothelial cells (ECs) of the heart proliferate and form new vessels in response to vascular endothelial growth factor (VEGF), but VEGF has not benefited the therapy of cardiac ischemia because of its side effects. Here, we explored if deletion of the vascular steady-state homeostasis maintaining Tie1 and Tie2 receptor tyrosine kinases affects the proliferation and sprouting of cardiac ECs. We analyzed EC proliferation and histological and immunohistochemical stainings by confocal microscopy, plus scRNA and qPCR analyses of gene expression in the heart, kidneys, and lungs of Tie1fl/fl, Tie2fl/fl, and Tie1fl/fl;Tie2fl/fl mice, in which vascular endothelial cadherin-driven CreERT2 recombinase was used to delete Tie1, Tie2 or both receptors. These analyses were also performed in mice subjected to transverse aortic constriction (TAC). Boyden chamber assays were performed to assess the migration of cultured ECs in cultures with or without TIE receptor silencing. Genetic deletion of Tie1, Tie2, or Tie1/Tie2 in mice increased significantly the proliferation of cardiac but not renal or pulmonary ECs, as measured by EdU incorporation into DNA and quantification of the cell cycle marker cyclin D1. Tie1/Tie2 or Tie2 deletion, but not Tie1 deletion alone, induced EC sprouting in coronary vasculature and expression of endothelial tip cell markers, including expression of the FOXO1-regulated Angpt2 and Esm1 genes in cardiac versus kidney or lung ECs. Consistent with these findings, silencing of TIE2, but not TIE1, in cultured ECs resulted in increased migration of ECs. Similar results were obtained in mice subjected to TAC. Deletion of Tie2 alone or together with Tie1 increases the proliferation and sprouting of cardiac, but not renal or pulmonary ECs, without to neovessel formation in the heart.

Abstract Linking glioblastoma (GBM) evolution to clinical progression is challenged by multiple factors, including tumor location for repeated sample collection, and short patient survival. In a single individual, we collected and analysed samples from 11 operations distributed across 31 months of multi-relapsing and multifocal GBM, including terminal leptomeningeal progression. All samples shared genomic ancestry of the retinoblastoma protein 1 (RB1) and neurofibromin 1 (NF1) mutations while advanced progression and extracranial metastases featured mutations of tuberous sclerosis complex 2 (TSC2) , PBRM1 , CD22 and Fanconi anemia supplementation group I ( FANCI ), correlated with clinical resistance to immunotherapies and DNA-damaging agents. Single-cell analytics revealed distinct yet reversible shifts in response to the precision medicine arsenal. GBM parenchymal dissemination and extracranial progression were associated with strengthening of neuron-like cell phenotypes. Our multidimensional study describes GBM evolution over a never reported time scale, and provides a valuable resource linking genetic, molecular, cellular and clinical progressions. Statement of significance: We assembled multidimensional omics data of mutlirelapsing GBM uncovering the cascade of mutations associated with GBM progression, transcriptomic response to therapies and evolution of phenotypic cell states that ultimately lead to extracranial progression. This unique resource sheds light on GBM evolution at the genetic, transcriptomic, cellular and clinical levels.

Abstract Retinopathy is a common symptom in mitochondrial diseases, and a leading cause of blindness in working-age individuals, often arising as a consequence of diabetes. Here, we demonstrate that postnatal loss of the replicative helicase of mitochondrial DNA in the astrocytes and Müller glia induces neovascular retinopathy. In these retinas, the macroglia show pathological reactivation, leading to hallmark features of neovascularization with blood-retina-barrier leakage, secondary microgliosis, and complement cascade activation. Similar reactivation of astrocytes in the cerebral cortex does not compromise vascular integrity, indicating tissue-specific roles of mitochondrial metabolism in macroglia for vascular homeostasis. Three secreted angiogenic factors—Fgf2, Pgf, and Lcn2—known to contribute to diabetic retinopathy, were induced. Spike recordings of the most sensitive retinal ganglion cells revealed normal rod function and intact retinal coding. These findings highlight the critical role of glial mitochondrial metabolism in neovascular retinopathy, with important implications for therapy development for mitochondrial and common forms of vision loss.

Obesity-induced changes in adipose vasculature are well documented, but systematic analyses in other tissues have not been as extensive. Here, we analyze the vessel-covered area and endothelial cell (EC) numbers in seven non-adipose organs (liver, heart, intestine, kidney, lung, ear skin, retina) in male mice after short-term high-fat diet (HFD) feeding (5 days or 5 weeks), long-term HFD feeding (10 weeks), and weight loss (5 weeks of HFD followed by 5 weeks of chow feeding). We show HFD-induced morphological changes in the liver, heart, kidney, and intestinal vasculatures, and a negative correlation between body weight and vessel density in most of the analyzed tissues. Interestingly, changes in vessel area do not always reflect alterations in EC numbers. Additionally, both the intestine and ear skin show preserved vessel perfusion in response to obesity. This study provides a comprehensive analysis of how different HFD feeding durations affect organotypic vasculature and reports that HFD feeding induces organ-specific vascular remodeling with distinct temporal dynamics in mice.

Glioblastoma progression features extensive dissemination of cancer cells throughout the brain. Limited by neuroimaging resolution and lack of specific markers, malignant cell infiltration is clinically undetectable. This makes complete tumor removal impossible and relapse inevitable. During their exodus from the tumor core, glioblastoma cells are traversing distinct brain territories influencing their phenotypic cell state. To better understand the cellular and molecular dynamics of glioblastoma invasion, we profiled glioblastoma patient avatars and clinical samples using total, single-cell and spatial transcriptomics. In addition to gene expression data, active intracellular pathways of glioblastoma invasion were screened with high throughput activated kinase assays (Pamgene). We identified that human glioblastoma stem cells exhibiting proneural (PN) and neuron-progenitor like (NPC) transcriptomic traits in vitro formed highly infiltrative and connected xenografts in patient avatars. Consistent with this, PN/NPC glioblastoma stem cells cultured on primary hippocampal neurons exhibited higher connectivity and motility compared to mesenchymal-like cells. Leveraging single-cell and spatial transcriptome analytics of avatar and patient samples, we performed cell clustering, annotation, pseudotime and RNA velocity analyses to associate glioblastoma phenotypic cell states to cellular functions. We found that invasive glioblastoma cells re-activated genes and pathways related to neuron progenitor maturation when transitioning from the tumor core to the brain microenvironment. To verify whether invasive PN/NPC glioblastoma cell integration would alter brain functionality, we performed preclinical live cerebral blood volume recordings using noninvasive brain functional ultrasounds (fUS). As glioblastomas progressed, we detected a gradual alteration of brain functional connectivity. Modifications happened in brain areas featuring minimal, yet consistent glioblastoma cell colonization, identified by post-mortem pathology analyses. Altogether, our data shed light on the importance of glioblastoma cell adaptability and brain integration during the metastatic progression. They also provide a framework for detecting invisible clinical features such as tumor invasion, focusing on brain functional connectivity assessments.

Glioblastomas (GBM) have the unique ability to hack neuronal plasticity. Neuroplasticity is controlled by brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB), for neuronal differentiation, survival and spinogenesis. To determine whether TrkB activity could pilot GBM cell fates, we screened total, single cell and spatial transcriptomics data from patient-derived glioblastoma stem cells (GSCs), preclinical and human samples. In parallel we used a curated library of drugs targeting TrkB including antidepressants and psychedelics, to control GBM cell fate. In silico: we generated total, single-cell and spatial transcriptomics data from mesenchymal (MES) and proneural (PN) patient-derived GSCs (N=10) and their in vivo analogues (N=9). In those, BDNF and TrkB gene expression and histological localization was verified and compared to published patient datasets (N=525). In vitro: on MES and PN GSCs, we verified TrkB and BDNF protein levels. We tested acute and chronic treatments with BDNF and/or five drugs targeting TrkB. Following treatments, GSC viability and sensitivity to temozolomide (TMZ) was quantified and tumor cell morphology analysed. In GSCs, preclinical and clinical dataset, we identified TrkB upregulation in PN subtypes. BDNF was expressed in the tumor-adjacent brain tissue but not in GBM cells. In all GSCs, BDNF treatment increased proliferation and cell morphology complexity. To determine whether TrkB activation drove PN GSC differentiation, we used pharmacological modulators of TrkB. We identified lisuride (LSD analogue) increased PN GSCs proliferation, interfered with cell adhesion and reduced TMZ cytotoxicity. Conversely, lisuride increased TMZ cytotoxicity in MES GSC, indicating subtype-specific phenotypic responses. These results provide a proof-of-concept for employing drugs targeting TrkB to harness GBM cell fate and chemosensitivity to TMZ. Such approaches may offer new avenues for combinatorial therapies targeting both GBM and its neural niche.

Despite strong indications that interactions between melanoma and lymphatic vessels actively promote melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk, we established human primary lymphatic endothelial cell (LEC) cocultures with human melanoma cell lines. Here, we show that coculture with melanoma cells induced transcriptomic changes in LECs and led to multiple changes in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found to contribute to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the coculture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC coculture escaped efficiently from the primary site to the proximal tumor-draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3/WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bidirectional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

Discovery of meningeal lymphatic vessels (LVs) in the dura mater, also known as dural LVs (dLVs) that depend on vascular endothelial growth factor C expression, has raised interest in their possible involvement in Alzheimer’s disease (AD). Here we find that in the APdE9 and 5xFAD mouse models of AD, dural amyloid-β (Aβ) is confined to blood vessels and dLV morphology or function is not altered. The induction of sustained dLV atrophy or hyperplasia in the AD mice by blocking or overexpressing vascular endothelial growth factor C, impaired or improved, respectively, macromolecular cerebrospinal fluid (CSF) drainage to cervical lymph nodes. Yet, sustained manipulation of dLVs did not significantly alter the overall brain Aβ plaque load. Moreover, dLV atrophy did not alter the behavioral phenotypes of the AD mice, but it improved CSF-to-blood drainage. Our results indicate that sustained dLV manipulation does not affect Aβ deposition in the brain and that compensatory mechanisms promote CSF clearance.

ABSTRACT Objective Secondary lymphedema (LE) following breast cancer surgery is a life-long complication, which currently has no cure. LE induces significant regional adipose tissue deposition, requiring liposuction as a treatment. Here, we aimed to elucidate the transcriptional, metabolomic, and lipidomic signature of the adipose tissue developed due to the surgery-induced LE in short- and long-term LE patients, and compared the transcriptomic landscape in LE to the obesity-induced adipose tissue. Methods Adipose tissue biopsies were obtained from breast cancer-operated females with LE from the affected and non-affected arms (n=20 patients). To decipher molecular properties of the LE adipose tissue, we performed RNA sequencing, metabolomics, and lipidomics combined with bioinformatics analyses. Results Integrative analysis of functional genomics revealed that inflammatory response, cell chemotaxis and angiogenesis were upregulated biological processes in the LE arm, indicating a sustained inflammation in the edematous adipose tissue, whereas, epidermal differentiation, cell-cell junction organization, water homeostasis and neurogenesis were, in turn, downregulated in the LE arm. Surprisingly, only few genes were found to be the same in the LE-induced and the obesity-induced adipose tissue expansion, indicating a different type of adipose tissue development in these two diseases. In metabolomics analysis, the concentration of a branched-chain amino acid valine was found to be reduced in the edematous arm together with downregulation of mRNA levels of its transporter SLC6A15 . Lipidomics analyses did not show any significant differences between the diseased and healthy arm, suggesting that diet affects the lipid composition of the adipose tissue more than the LE. Conclusions Our results provide a detailed molecular characterization of adipose tissue in secondary LE of breast cancer patients vs individuals with obesity. The results show distinct differences in transcriptomic signatures between LE patients vs individuals with obesity, but only minor differences in metabolome and lipidome between the diseased and the healthy arm.

<p>PDF - 75K, Levels of macrophage- and macrophage polarizationassociated cytokines.</p>

ABSTRACT Despite strong indications that melanoma interaction with lymphatic vessels actively promotes melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk we established human primary lymphatic endothelial cell (LEC) co-cultures with human melanoma cell lines. Here, we show that co-culture with melanoma cells induced transcriptomic changes in LECs and led to multiple alterations in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found contributing to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the co-culture with LECs, and Notch3 and WNT5B were co-expressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC co-culture escaped efficiently from the primary site to the proximal tumor draining lymph nodes, which was impaired upon WNT5B depletion. This supports the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3-WNT5B axis in melanoma. This study elucidates WNT5B as a novel molecular factor mediating bi-directional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

PDF - 1798K, Blood leukocyte and monocyte counts.

Macrophage Clever-1 contributes to impaired antigen presentation and suppression of anti-tumor immunity. This first-in-human trial investigates the safety and tolerability of Clever-1 blockade with bexmarilimab in patients with treatment-refractory solid tumors and assesses preliminary anti-tumor efficacy, pharmacodynamics, and immunologic correlates. Bexmarilimab shows no dose-limiting toxicities in part I (n = 30) and no additional safety signals in part II (n = 108). Disease control (DC) rates of 25%-40% are observed in cutaneous melanoma, gastric, hepatocellular, estrogen receptor-positive breast, and biliary tract cancers. DC associates with improved survival in a landmark analysis and correlates with high pre-treatment intratumoral Clever-1 positivity and increasing on-treatment serum interferon γ (IFNγ) levels. Spatial transcriptomics profiling of DC and non-DC tumors demonstrates bexmarilimab-induced macrophage activation and stimulation of IFNγ and T cell receptor signaling selectively in DC patients. These data suggest that bexmarilimab therapy is well tolerated and show that macrophage targeting can promote immune activation and tumor control in late-stage cancer.

The small intestine is an excellent model for studying changes in vasculature in response to different diseases or gene deletions. Here, we present a protocol for whole-mount immunofluorescence staining of blood and lymphatic vessels in the adult mouse small intestine. We describe the steps for perfusion fixation, tissue sample preparation, immunofluorescence staining, and whole-mount preparation of stained samples. Our protocol will enable researchers to visualize and analyze the intricate network of vessels in the small intestine.

PDF - 932K, Immunofluorescence staining of CD45+,CD4+ and CD45+,CD8+ T cells in DMBA-TPA treated skin of wild-type, control transgenic and transgenic mice.

The recent discovery of lymphatic vessels (LVs) in the dura mater, the outermost layer of meninges around the central nervous system (CNS), has opened a possibility for the development of alternative therapeutics for CNS disorders. The vascular endothelial growth factor C (VEGF-C)/VEGF receptor 3 (VEGFR3) signaling pathway is essential for the development and maintenance of dural LVs. However, its significance in mediating dural lymphatic function in CNS autoimmunity is unclear. We show that inhibition of the VEGF-C/VEGFR3 signaling pathway using a monoclonal VEGFR3-blocking antibody, a soluble VEGF-C/D trap, or deletion of the Vegfr3 gene in adult lymphatic endothelium causes notable regression and functional impairment of dural LVs but has no effect on the development of CNS autoimmunity in mice. During autoimmune neuroinflammation, the dura mater was only minimally affected, and neuroinflammation-induced helper T (T H ) cell recruitment, activation, and polarization were significantly less pronounced in the dura mater than in the CNS. In support of this notion, during autoimmune neuroinflammation, blood vascular endothelial cells in the cranial and spinal dura expressed lower levels of cell adhesion molecules and chemokines, and antigen-presenting cells (i.e., macrophages and dendritic cells) had lower expression of chemokines, MHC class II–associated molecules, and costimulatory molecules than their counterparts in the brain and spinal cord, respectively. The significantly weaker T H cell responses in the dura mater may explain why dural LVs do not contribute directly to CNS autoimmunity.

PDF - 1898K, Expression of neuropilins in skin and tumors of wild-type mice.