The omentum, a specialized adipose tissue within the peritoneum, is a primary niche of ovarian cancer dissemination. Omental adipocytes are widely thought to promote tumor growth by supplying lipids, supported in part by studies using global FABP4 deficiency. Here, we directly test whether mature adipocytes are required for peritoneal ovarian cancer growth using mice congenitally lacking mature adipocytes within the peritoneal cavity, including the omentum. Across several ovarian cancer models (ID8p53−/−Brca2−/−, BPPNM, and KPCA), tumors preferentially seed adipose-associated regions despite the absence of mature adipocytes. Loss of mature adipocytes does not impair peritoneal tumor expansion, whereas removal of the adipocyte-free omentum significantly reduces tumor burden. Murine and human single-cell transcriptomic analyses reveal enrichment of lipid-handling gene expression, including FABP4, in omental endothelial cells at steady state and in tumor-bearing tissue. Endothelial-specific deletion of FABP4 reduces omental tumor expansion and limits tumor vascular complexity. These findings indicate that mature adipocytes are not required for omental ovarian cancer growth and highlight tumor-growth-favoring features of the omental niche endothelium. Ovarian cancer (OC) often spreads to the omentum, an adipose-rich tissue in the abdomen. Here, authors show that FABP4-expressing endothelial cells contribute to OC growth in the omentum independently of mature adipocytes.

Accumulating evidence demonstrates that the central nervous system (CNS) is not disconnected from the peripheral immune system; however, precisely how the adaptive immune system surveils the CNS remains a critical question. Recent findings reveal that channels between the skull and the dura mater facilitate the exchange of cerebrospinal fluid and immune cells between the CNS and skull bone marrow of mice under both homeostatic and disease conditions1-6. Skull bone marrow functions as a source of immune cells for the CNS5, yet its role in CNS antigen-specific adaptive immune responses remains unclear. Here we identify lymphoid structures within the skull bone marrow, featuring germinal-centre-like formations and containing a distinct population of follicular-helper-like T cells that promote B cell activation and humoral immunity through CD40L, IL-21 and IFNγ signalling. Adaptive immune cells within these skull bone marrow lymphoid structures surveil and respond to CNS-derived antigens and contribute to anti-tumour immune responses in mouse brain cancer models. Together, our findings show that the skull bone marrow is a site of CNS immunosurveillance that may influence immune responses across diverse neurological diseases.

BACKGROUND & AIMS: Short bowel syndrome (SBS) arises from the surgical removal of extensive portions of the small intestine and is associated with high morbidity, including intestinal failure-associated liver disease (IFALD). Earlier studies revealed that orally administered systemic liver X receptor (LXR) agonist suppresses IFALD in mice and implicated intestinally derived high-density lipoprotein (HDL) in liver protection. Here we aimed to move away from the use of systemic LXR agonists because they have failed in clinical trials due to hepatic steatosis and hyperlipidemia, to determine if a gut-restricted LXR agonist could provide hepatoprotection in SBS. METHODS: We synthesized and characterized WUSTL0717, an amide analog of GW3965, as a putative gut-restricted LXR agonist, and evaluated its potential to improve the outcomes in a preclinical mouse model of SBS. RESULTS: WUSTL0717 exhibited exceptional intestinal retention in pharmacokinetic analyses and activated LXR target genes in the small intestine but not the liver. Whereas small bowel resection lowered many lipid metabolites in portal venous plasma, WUSTL0717 treatment increased portal venous Apolipoprotein A1 (ApoA1), the core protein of HDL, and spared portal venous phospholipids known to be enriched on HDL. Accordingly, intestinal ApoA1 deficiency exacerbated IFALD, and in wild-type mice, portal venous ApoA1 and phospholipids inversely correlated with hepatic collagen accumulation. In addition, WUSTL0717 improved nutrient absorption and promoted body weight restoration in SBS. CONCLUSIONS: These data underscore the potential of gut-restricted LXR agonists to preserve metabolic health in the context of SBS. By acting locally in the intestine, WUSTL0717 positively mitigates profibrotic liver injury while avoiding systemic availability.

Peritoneal cavity fluid and mesothelial surfaces host distinct resident macrophage populations, among which include the well-described Gata6+ large cavity macrophages (LCMs) in peritoneal fluid. Here, we reveal that LCMs arise from two separable differentiation pathways. In the quantitatively minor pathway, monocytes gave rise to LYVE1+ LCMs but few Gata6+ LCMs. This pathway did not require the transcription factor Gata6 but was severely impaired in mice bearing three mutations in the -165 kb Zeb2 enhancer (Zeb2TM) with impaired monocyte development. The second, dominant pathway supported Gata6-dependent LCMs and was intact in Zeb2TM mice, even when turnover was enforced by irradiation, and was supported by adoptive transfer of a specialized LCM intermediate expressing Gata6 before the residency marker TIMD4. Functionally, the quantitatively minor LCM pathway distinctly surveilled the mesothelium, replenishing mesothelial border macrophages upon encountering an open niche. Thus, beyond embryonic versus adult hematopoietic paradigms, LCMs with overlapping and distinct phenotypes arise from two pathways linked to divergent fates.

The pathophysiology of ulcerative colitis (UC) is driven by a disrupted intestinal barrier that triggers aberrant immune responses to gut commensal microbes. The lymphatic vasculature is critical for maintaining intestinal immune homeostasis, and lymphatic expansion is a hallmark of UC pathology. However, the molecular drivers and functional consequences of increased lymphangiogenesis remain unclear. During development, PROX1+/LYVE1+ lymphatic endothelial cell (LEC) progenitors originating from the cardinal vein form a mature lymphatic network through sprouting lymphangiogenesis. We recently showed that lymphatic vessels in the colonic mucosa run along the base of the crypts, extending into individual mucosal folds, and begin developing shortly after birth. High resolution imaging of colons from newborn pups revealed PROX1+/LYVE1+ mesenteric lymphatic vessels beginning to invade the wall of the colon and several individual PROX1+/LYVE1- LECs dispersed throughout the mucosa. By day 2, these LECs appeared near the growing mucosal fold lymphatics extending from the mesentery. These data suggest that the colon LECs are heterogenous in origin and we hypothesize that the distinct LEC populations exhibit unique spatial patterns, gene expression and functions in the colon. This study aims to describe the functional consequences of colonic LEC heterogeneity under homeostatic and inflammatory conditions with the goal of identifying new therapeutic targets for treatment of UC. NIH NIAD grant U01AI163064 Mucosal and Regional Immunology (MUC)

Mouse resident peritoneal macrophages, called large cavity macrophages (LCM), arise from embryonic progenitors that proliferate as mature, CD73+Gata6+ tissue-specialized macrophages. After injury from irradiation or inflammation, monocytes are thought to replenish CD73+Gata6+ LCMs through a CD73-LYVE1+ LCM intermediate. Here, we show that CD73-LYVE1+ LCMs indeed yield Gata6+CD73+ LCMs through integrin-mediated interactions with mesothelial surfaces. CD73-LYVE1+ LCM repopulation of the peritoneum was reliant upon and quantitatively proportional to recruited monocytes. Unexpectedly, fate mapping indicated that only ~10% of Gata6-dependent LCMs that repopulated the peritoneum after injury depended on the LYVE1+ LCM stage. Further supporting nonoverlapping lifecycles of CD73-LYVE1+ and CD73+Gata6+ LCMs, in mice bearing a paucity of monocytes, Gata6+CD73+ LCMs rebounded after ablative irradiation substantially more efficiently than their presumed LYVE1+ or CD73- LCM upstream precursors. Thus, after inflammatory insult, two temporally parallel pathways, each generating distinct differentiation intermediates with varying dependencies on monocytes, contribute to the replenish hment of Gata6+ resident peritoneal macrophages.

Light sheet microscopy and preparative clearing methods that improve light penetration in 3D tissues have revolutionized imaging in biomedical research. Here we present ADAPT-3D, a streamlined 3-step approach to turn tissues optically transparent while preserving tissue architecture with the versatility to handle diverse tissue sizes and types across species. Unlike extensive lipid removal utilized by existing protocols, ADAPT-3D only partially removes lipids to preserve cell membranes, yet the non-toxic aqueous refractive indexing solution still rapidly turns tissues transparent while preserving the fluorescence of endogenous and antibody conjugated fluorophores. ADAPT-3D prepares whole mouse brains for light sheet microscopy in a 4-hour refractive indexing step after less than 4 days of preprocessing without changing their size. By maintaining tissue size, ADAPT-3D clears 1-mm thick brain slices in under 24 h without causing damage and facilitates a 3D section-like view of the meandering choroid plexus. We applied ADAPT-3D to overcome challenges of whole mouse skull clearing and visualized the undisturbed brain borders including specialized skull channels after just 8 days of tissue preparation. ADAPT-3D also had utility in clearing and immunolabeling human intestinal tissues in about 5 days. Overall, ADAPT-3D provides a high-speed, non-shrinking, and fluorescence-preserving workflow for 3D imaging that bridges section-based and whole-organ studies, offering new opportunities for biological discovery.

Supplementary Figures and Tables.

The omentum, a specialized adipose tissue within the peritoneum, is a primary niche for ovarian cancer (OC) dissemination during peritoneal carcinomatosis. Traditionally, omental adipocytes are thought to promote OC growth by supplying lipids, supported by evidence that global FABP4 deficiency reduces tumor progression. Here, we generated mice lacking mature adipocytes in the peritoneum, including the omentum. ID8p53-/-Brca2-/-, BPPNM, and KPCA OC cells retained a propensity to seed regions typically associated with adipocytes, even without mature adipocytes. However, the lack of mature adipocytes did not suppress peritoneal OC expansion, whereas removing the adipocyte-free omentum did. Murine and human single-cell RNA sequencing revealed that endothelial FABP4 was high in the omentum. Indeed, endothelial cell-selective deficiency of FABP4 reduced OC growth in the peritoneum. These findings prompt a reevaluation of adipocyte contributions to OC progression and suggest a key role of the omental vasculature in supporting OC metabolic growth.

Innovations in 3D tissue imaging have revolutionized research, but limitations stemming from lengthy protocols and equipment accessibility persist. Widefield microscopy is fast and accessible but often excluded from 3D imaging workflows due to its lack of optical sectioning. Here we combine tissue clearing with a commercial depth-variant deconvolution approach that we optimized for large-volume widefield imaging. By implementing prefiltering with z-brick splitting, we achieve subnuclear axial resolution in tissues to a depth of 500 µm in multi-tile scan images. We illustrate the utility of this method in a model of ileitis and to gain a 3D perspective in thick brain slices from a model of cerebral amyloid angiopathy, where we resolve amyloid deposits along small blood vessels, attaining resolution that compared favorably to confocal microscopy. Finally, we leverage our approach to image hundreds of consecutive z planes for richer evaluation of cleared human kidney biopsies in a simulated, transplant time window and visualized atrophic tubules and winding arterioles associated with glomeruli in 3D. Having achieved subnuclear z-resolution in sections hundreds of microns thick, coupling widefield microscopy of cleared tissue to robust deconvolution now emerges as an accessible and viable method to gain 3D insight in research or clinical evaluations.

The colon is characterized by histological undulations and posited to play a role in fecal formation through water absorption. While human colons exhibit haustral folds along with less pronounced intrahaustral folds, murine colons are devoid of these characteristics yet demonstrate a consistent pattern of luminal folds, particularly within the proximal region. Our advanced intravital and three-dimensional imaging techniques applied across murine and human tissues have revealed a novel function of these folds: operating for managing interstitial fluid, facilitated by a specialized lymphatic network anchored into the folds. These folds are crucial to colonic lymphatic drainage, functioning as conduits for fluid outflow and immune surveillance. The presence of lymphoid follicles and phagocytic macrophages within this system reflects an advanced role in immune surveillance, in contrast to the uniform lymphatic flow observed in the small intestine. In mice, colitis appears to compromise the structural integrity of these folds, resulting in the propagation of inflammation from distal to proximal regions, which parallels patterns seen in human ulcerative colitis, suggesting that the integrity of colonic folds may be vital to understanding the progression of IBD. In conclusion, our research posits the colonic folds as essential components in fluid dynamics and immune regulation, yielding new insights into the pathogenesis of IBD and potential therapeutic strategies. NIH DP1DK109668 NIH R01AI168044 NIH U01AI163064 Crohn’s & Colitis Foundation Career Developmental Award #938100 Medical Research Council Clinical Research Training Fellowship Society for Mucosal Immunology Technique-Sharing Grant Mucosal and Regional Immunology (MUC)

Abstract More than 80% of ovarian cancer (OC) cases have metastasized to the peritoneal cavity at diagnosis. The five-year survival rate for these patients is 25%. Within the peritoneal cavity, the most common and early site of metastasis is the omentum, a well-vascularized, specialized adipose tissue that arises off the greater curvature of the stomach. Because OC homes to the omentum, it is removed as a standard of care for patients with peritoneal metastases. In mouse models, the omentum is also an early metastatic site, and omentectomy reduces tumor expansion. The mechanism by which the omentum promotes OC growth is unknown, perhaps due to the complex omental microenvironment. The omentum contains many features that could impact OC seeding. It is covered by a mesothelial lining that extends in a sheet anchoring the omentum to surrounding tissues and is highly perfused by blood vessels. It contains lymphoid aggregates or milky spots that may contribute to pro- and anti-tumor immunity. This study explores the association of OC metastases with specific omentum features. We intraperitoneally injected luciferase-expressing ID8 OC cells to mimic high-grade serous OC metastasis. Using high-resolution, spatial ex vivo bioluminescence imaging (BLI), we found that tumor nodules preferentially formed in adipocyte-rich regions of the omentum and the mesentery, another peritoneal tissue favorable for OC seeding. To determine whether adipocytes, the most abundant omental cell type, are the primary support of OC, we crossed Adiponectin Cre+ and DTA mice to generate mice lacking white and brown adipocytes from birth. To overcome the metabolic syndrome the fatless mice develop, they receive subcutaneous fat transplants dorsally, referred to as distal adipocyte rescue of lipodystrophy (DARL) mice. Littermate controls receive sham surgeries. The blood triglyceride, glucose, and insulin levels are normalized post-transplant. Thus, the peritoneal cavity lacks mature adipocytes, but the mice have typical metabolic profiles, allowing us to study the role of omental adipocytes in peritoneal metastasis. Once the mice recovered from surgery, they were intraperitoneally injected with luciferase-expressing ID8s. While it was hypothesized that OC metastatic seeding within the adipocyte-free omentum and associated peritoneum would be impaired without local mature adipocytes, BLI revealed that the tumor burden was not decreased in DARL mice. Notably, specific regions of OC seeding, where mature adipocytes would have localized if present, such as the omentum and mesentery, retained tumor nodules in DARL mice. This conserved OC localization suggests that other cells in this specific microenvironment might be responsible for metastatic spread. To identify these cells, studies are ongoing to profile these regions in the DARL and control mice via flow cytometry and single-cell RNA sequencing. By unraveling the support for OC cells in the omentum, therapeutic strategies can be identified to repolarize the metastatic milieu and halt further spread to improve patient outcomes. Citation Format: Rachel Mintz, Alexandre Gallerand, Jichang Han, Sarah Ning, Wei Zou, Mary Wohltmann, Bernd Zinselmeyer, Gwendalyn Randolph. Mechanisms underlying the omental support of ovarian cancer peritoneal metastasis [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B091.

Extramedullary hematopoiesis (EMH) expands hematopoietic capacity outside of the bone marrow in response to inflammatory conditions, including infections and cancer. Because of its inducible nature, EMH offers a unique opportunity to study the interaction between hematopoietic stem and progenitor cells (HSPCs) and their niche. In cancer patients, the spleen frequently serves as an EMH organ and provides myeloid cells that may worsen pathology. Here, we examined the relationship between HSPCs and their splenic niche in EMH in a mouse breast cancer model. We identify tumor produced IL-1α and leukemia inhibitory factor (LIF) acting on splenic HSPCs and splenic niche cells, respectively. IL-1α induced TNFα expression in splenic HSPCs, which then activated splenic niche activity, while LIF induced proliferation of splenic niche cells. IL-1α and LIF display cooperative effects in activating EMH and are both up-regulated in some human cancers. Together, these data expand avenues for developing niche-directed therapies and further exploring EMH accompanying inflammatory pathologies like cancer.

The pore-forming S. aureus a-toxin (Hla) contributes to virulence and disease pathogenesis. While high concentrations of toxin induce cell death, neutrophils exhibit relative resistance to lysis, suggesting that the action of Hla may not be solely conferred by lytic susceptibility. Using intravital microscopy, we observed that Hla disrupts neutrophil localization and clustering early in infection. Hla forms a narrow, ion-selective pore, suggesting that Hla may dysregulate calcium or other ions to impair neutrophil function. We found that sub-lytic Hla did not permit calcium influx but caused rapid membrane depolarization. Depolarization decreases the electrogenic driving force for calcium, and concordantly, Hla suppressed calcium signaling in vitro and in vivo and calcium-dependent leukotriene B4 (LTB4) production, a key mediator of neutrophil clustering. Thus, Hla disrupts the early patterning of the neutrophil response to infection, in part through direct impairment of neutrophil calcium signaling. This early mis-localization of neutrophils may contribute to establishment of infection.

Abstract The central nervous system (CNS) antigen-presenting cell (APC) that primes antitumor CD8+ T-cell responses remains undefined. Elsewhere in the body, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain parenchyma cDC1 are extremely rare; cDCs localize to the choroid plexus and dura. Thus, whether the cDC1 play a function in presenting antigen derived from parenchymal sources in the tumor setting remains unknown. Using preclinical glioblastoma (GBM) models and cDC1-deficient mice, we explored the presently unknown role of cDC1 in CNS antitumor immunity. We determined that, in addition to infiltrating the brain tumor parenchyma itself, cDC1 prime neoantigen-specific CD8+ T cells against brain tumors and mediate checkpoint blockade-induced survival benefit. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1 equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same APC subsets, we identified a tumor-specific fluorescent metabolite of 5-aminolevulinic acid, which fluorescently labeled tumor cells during fluorescence-guided GBM resection. Together, these data elucidate the specialized behavior of cDC1 and suggest that cDC1 play a significant role in CNS antitumor immunity.

Recently, studies have emerged suggesting that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. We investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. Na+ accumulation was induced in rats by a high salt diet (HSD) (8% NaCl and 1% saline to drink) or by implantation of a deoxycorticosterone acetate (DOCA) tablet (1% saline to drink) using rats on a low salt diet (LSD) (0.1% NaCl) on tap water as control. Na+ and K+ were assessed by ion chromatography in tissue eluates, and the extracellular volume by equilibration of Cr-51-EDTA. By tangential sectioning of the skin, we found a low Na+ content and extracellular volume in epidermis, both parameters rising by similar to 30% and 100%, respectively, in LSD and even more in HSD and DOCA when entering dermis. We found evidence for an extracellular Na+ gradient from epidermis to dermis shown by an estimated concentration in epidermis similar to 2 and 4-5 times that of dermis in HSD and DOCA-salt. There was intracellular storage of Na+ in skin, muscle, and myocardium without a concomitant increase in hydration. Our data suggest that there is a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. Salt stress results in intracellular storage of Na+ in exchange with K+ in skeletal muscle and myocardium that may have electromechanical consequences. Key points Studies have suggested that Na+ can be retained or removed without commensurate water retention or loss, and that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. In the present study, we investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. We used two common models for salt-sensitive hypertension: high salt and a deoxycorticosterone salt diet. We found a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. There was intracellular Na+ storage in muscle and myocardium without a concomitant increase in hydration, comprising storage that may have electromechanical consequences in salt stress.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

Two resident macrophage subsets reside in peritoneal fluid. Macrophages also reside within mesothelial membranes lining the peritoneal cavity, but they remain poorly characterized. Here, we identified two macrophage populations (LYVE1hi MHC IIlo-hi CX3CR1gfplo/− and LYVE1lo/− MHC IIhi CX3CR1gfphi subsets) in the mesenteric and parietal mesothelial linings of the peritoneum. These macrophages resembled LYVE1+ macrophages within surface membranes of numerous organs. Fate-mapping approaches and analysis of newborn mice showed that LYVE1hi macrophages predominantly originated from embryonic-derived progenitors and were controlled by CSF1 made by Wt1+ stromal cells. Their gene expression profile closely overlapped with ovarian tumor-associated macrophages previously described in the omentum. Indeed, syngeneic epithelial ovarian tumor growth was strongly reduced following in vivo ablation of LYVE1hi macrophages, including in mice that received omentectomy to dissociate the role from omental macrophages. These data reveal that the peritoneal compartment contains at least four resident macrophage populations and that LYVE1hi mesothelial macrophages drive tumor growth independently of the omentum.