Scientific Resources

OBJECTIVE The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa),this phenotype is reprogrammed,making it an interesting therapeutic target. However,how the truncated prostate TCA cycle works is still not completely understood. METHODS We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis. RESULTS First,mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate,thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis,several key nutrients were shown to allow the completion of the prostate TCA cycle,revealing a much more complex metabolic profile than originally anticipated. Indeed,along with the known pathway of aspartate replenishing oxaloacetate,glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids,aspartate entered the TCA cycle at the malate entry point,upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate-oxaloacetate-citrate pathway,but also by at least three additional pathways: glutaminolysis,reductive carboxylation,and aspartate-malate conversion. CONCLUSIONS Our results add a significant new dimension to the prostate citrate-secretory phenotype,with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa. View Publication

Fibroblasts are the most abundant stromal constituents of the tumour microenvironment in primary as well as metastatic colorectal cancer (CRC). Their supportive effect on tumour cells is well established. There is growing evidence that stromal fibroblasts also modulate the immune microenvironment in tumours. Here,we demonstrate a difference in fibroblast-mediated immune modulation between primary CRC and peritoneal metastasis. Cancer-associated fibroblasts (CAFs) were isolated from primary cancer and from peritoneal metastases (MAFs) from a total of 17 patients. The ectoenzyme CD38 was consistently expressed on the surface of all MAFs,while it was absent from CAFs. Furthermore,MAFs secreted higher levels of IGFBP2,CXCL2,CXCL6,CXCL12,PDGF-AA,FGFb,and IL-6. This was associated with a decreased activation of macrophages and a suppression of CD25 expression and proliferation of co-cultivated T-cells. Downregulation of IGFBP2 abolished these immunosuppressive effects of MAFs. Taken together,these results show that MAFs contribute to an immunosuppressive tumour microenvironment in CRC metastases by modulating the phenotype of immune cells through an IGFBP2-dependent mechanism. View Publication

BACKGROUND Hypoxia and inflammation tumor microenvironment (TME) play a crucial role in tumor development and progression. Although increased understanding of TME contributed to gastric cancer (GC) progression and prognosis,the direct interaction between macrophage and GC cells was not fully understood. METHODS Hypoxia and normoxia macrophage microarrays of GEO database was analyzed. The peripheral blood mononuclear cell acquired from the healthy volunteers. The expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) in GC tissues and cell lines was detected by quantitative reverse transcription PCR (qRT-PCR),western-blot,Elisa and immunofluorescence. Cell proliferation,migration,and invasion were evaluated by cell counting kit 8 (CCK8),colony formation,real-time imaging of cell migration and transwell. Flow Cytometers was applied to identify the source of cytokines. Luciferase reporter assays and chromatin immunoprecipitation were used to identify the interaction between transcription factor and target gene. Especially,a series of truncated and mutation reporter genes were applied to identify precise binding sites. The corresponding functions were verified in the complementation test and in vivo animal experiment. RESULTS Our results revealed that hypoxia triggered macrophage secreted CXCL8,which induced GC invasion and proliferation. This macrophage-induced GC progression was CXCL8 activated C-X-C Motif Chemokine Receptor 1/2 (CXCR1/2) on the GC cell membrane subsequently hyperactivated Janus kinase 1/ Signal transducer and activator of transcription 1 (JAK/STAT1) signaling pathway. Then,the transcription factor STAT1 directly led to the overexpression and secretion of Interleukin 10 (IL-10). Correspondingly,IL-10 induced the M2-type polarization of macrophages and continued to increase the expression and secretion of CXCL8. It suggested a positive feedback loop between macrophage and GC. In clinical GC samples,increased CXCL8 predicted a patient's pessimistic outcome. CONCLUSION Our work identified a positive feedback loop governing cancer cells and macrophage in GC that contributed to tumor progression and patient outcome. View Publication

BACKGROUND CD47/SIRP$\alpha$ axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer,particularly in hematological malignancies. In preclinical models,CD47/SIRP$\alpha$ blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here,we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. METHODS The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry,transcriptomic analysis,and in vivo immune-cell depletion experiments. RESULTS We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells,monocytes,dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably,molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation,including IFN$\gamma$ and IL-12b. Moreover,TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701,highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition,NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro,resulting in an increased cross-priming of tumor-specific CD8 T cells. CONCLUSIONS The study described the mechanisms afforded by the CD47xCD19 bispecific antibody,NI-1701,at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035). View Publication

BACKGROUND Recent investigations of the meninges have highlighted the importance of the dura layer in central nervous system immune surveillance beyond a purely structural role. However,our understanding of the meninges largely stems from the use of pre-clinical models rather than human samples. METHODS Single-cell RNA sequencing of seven non-tumor-associated human dura samples and six primary meningioma tumor samples (4 matched and 2 non-matched) was performed. Cell type identities,gene expression profiles,and T cell receptor expression were analyzed. Copy number variant (CNV) analysis was performed to identify putative tumor cells and analyze intratumoral CNV heterogeneity. Immunohistochemistry and imaging mass cytometry was performed on selected samples to validate protein expression and reveal spatial localization of select protein markers. RESULTS In this study,we use single-cell RNA sequencing to perform the first characterization of both non-tumor-associated human dura and primary meningioma samples. First,we reveal a complex immune microenvironment in human dura that is transcriptionally distinct from that of meningioma. In addition,we characterize a functionally diverse and heterogenous landscape of non-immune cells including endothelial cells and fibroblasts. Through imaging mass cytometry,we highlight the spatial relationship among immune cell types and vasculature in non-tumor-associated dura. Utilizing T cell receptor sequencing,we show significant TCR overlap between matched dura and meningioma samples. Finally,we report copy number variant heterogeneity within our meningioma samples. CONCLUSIONS Our comprehensive investigation of both the immune and non-immune cellular landscapes of human dura and meningioma at single-cell resolution builds upon previously published data in murine models and provides new insight into previously uncharacterized roles of human dura. View Publication

Resistance mechanisms and heterogeneity in HER2-positive gastric cancers (GC) limit Trastuzumab benefit in 32% of patients,and other targeted therapies have failed in clinical trials. Using patient samples,patient-derived xenografts (PDXs),partially humanized biological models,and HER2-targeted imaging technologies we demonstrate the role of caveolin-1 (CAV1) as a complementary biomarker in GC selection for Trastuzumab therapy. In retrospective analyses of samples from patients enrolled on Trastuzumab trials,the CAV1-high profile associates with low membrane HER2 density and low patient survival. We show a negative correlation between CAV1 tumoral protein levels - a major protein of cholesterol-rich membrane domains - and Trastuzumab-drug conjugate TDM1 tumor uptake. Finally,CAV1 depletion using knockdown or pharmacologic approaches (statins) increases antibody drug efficacy in tumors with incomplete HER2 membranous reactivity. In support of these findings,background statin use in patients associates with enhanced antibody efficacy. Together,this work provides preclinical justification and clinical evidence that require prospective investigation of antibody drugs combined with statins to delay drug resistance in tumors. View Publication

Impaired T cell immunity with aging increases mortality from infectious disease. The branching of Asparagine-linked glycans is a critical negative regulator of T cell immunity. Here we show that branching increases with age in females more than males,in na{\{i}}ve more than memory T cells and in CD4+ more than CD8+ T cells. Female sex hormones and thymic output of na{\"{i}}ve T cells (TN) decrease with age however neither thymectomy nor ovariectomy altered branching. Interleukin-7 (IL-7) signaling was increased in old female more than male mouse TN cells and triggered increased branching. N-acetylglucosamine a rate-limiting metabolite for branching increased with age in humans and synergized with IL-7 to raise branching. Reversing elevated branching rejuvenated T cell function and reduced severity of Salmonella infection in old female mice. These data suggest sex-dimorphic antagonistic pleiotropy where IL-7 initially benefits immunity through TN maintenance but inhibits TN function by raising branching synergistically with age-dependent increases in N-acetylglucosamine." View Publication

Alveolar macrophages (AMs) are functionally important innate cells involved in lung homeostasis and immunity and whose diversity in health and disease is a subject of intense investigations. Yet,it remains unclear to what extent conditions like smoking or chronic obstructive pulmonary disease (COPD) trigger changes in the AM compartment. Here,we aimed to explore heterogeneity of human AMs isolated from healthy nonsmokers,smokers without COPD,and smokers with COPD by analyzing BAL fluid cells by flow cytometry and bulk and single-cell RNA sequencing. We found that subpopulations of BAL fluid CD206+ macrophages could be distinguished based on their degree of autofluorescence in each subject analyzed. CD206+ autofluorescenthigh AMs were identified as classical,self-proliferative AM,whereas autofluorescentlow AMs were expressing both monocyte and classical AM-related genes,supportive of a monocytic origin. Of note,monocyte-derived autofluorescentlow AMs exhibited a functionally distinct immunoregulatory profile,including the ability to secrete the immunosuppressive cytokine IL-10. Interestingly,single-cell RNA-sequencing analyses showed that transcriptionally distinct clusters of classical and monocyte-derived AM were uniquely enriched in smokers with and without COPD as compared with healthy nonsmokers. Of note,such smoking-associated clusters exhibited gene signatures enriched in detoxification,oxidative stress,and proinflammatory responses. Our study independently confirms previous reports supporting that monocyte-derived macrophages coexist with classical AM in the airways of healthy subjects and patients with COPD and identifies smoking-associated changes in the AM compartment that may favor COPD initiation or progression. View Publication

Interleukin-4 (IL-4) is a signature cytokine pivotal in Type 2 helper T cell (Th2) immune response,particularly in allergy and hypersensitivity. Interestingly,IL-4 increases endogenous levels of prostaglandin D2 (PGD2 ) and its metabolites,$\Delta$12 -prostaglandin J2 ($\Delta$12 -PGJ2 ) and 15-deoxy-$\Delta$12,14 -prostaglandin J2 (15d-PGJ2 ),collectively called cyclopentenone PGs (CyPGs). However,the therapeutic role of IL-4 in hematologic malignancies remains unclear. Here,we employed a murine model of acute myeloid leukemia (AML),where human MLL-AF9 fusion oncoprotein was expressed in hematopoietic progenitor cells,to test the effect of IL-4 treatment in vivo. Daily intraperitoneal treatment with IL-4 at 60 µg/kg/d significantly alleviated the severity of AML,as seen by decreased leukemia-initiating cells (LICs). The effect of IL-4 was mediated,in part,by the enhanced expression of hematopoietic- PGD2  synthase (H-PGDS) to effect endogenous production of CyPGs,through autocrine and paracrine signaling mechanisms. Similar results were seen with patient-derived AML cells cultured ex vivo with IL-4. Use of GW9662,a peroxisome proliferator-activated receptor gamma (PPAR$\gamma$) antagonist,suggested endogenous CyPGs-PPAR$\gamma$ axis mediated p53-dependent apoptosis of LICs by IL-4. Taken together,our results reveal a beneficial role of IL-4 treatment in AML suggesting a potential therapeutic regimen worthy of clinical trials in patients with AML. View Publication

Human norovirus (HNoV) is a global health and socioeconomic burden,estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole blood were infected with HNoV-positive stool samples and harvested at 3??days postinfection (dpi) to assess the viral RNA yield by reverse transcriptase quantitative PCR (RT-qPCR). A 3- to 18-fold increase in the HNoV RNA yield was observed in 50 to 60% of donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsy specimens. Next,we characterized infection of whole-blood-derived B cells by flow cytometry in specific functional B cell subsets (naive CD27- IgD+,memory-switched CD27+ IgD-,memory-unswitched CD27+ IgD+,and double-negative CD27- IgD- cells). While the susceptibilities of the subsets were similar,changes in the B cell subset distribution upon infection were observed,which were also noted after treatment with HNoV virus-like particles and the predicted recombinant NS1 protein. Importantly,primary B cell stimulation with the predicted recombinant NS1 protein triggered B cell activation and induced metabolic changes. These data demonstrate that primary B cells are susceptible to HNoV infection and suggest that the NS1 protein can alter B cell activation and metabolism in vitro,which could have implications for viral pathogenesis and immune responses in vivo. IMPORTANCE Human norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised,the elderly,and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socioeconomic burdens associated with HNoV infections. Moreover,HNoV does not elicit lifelong immunity as repeat infections are common,presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity,we investigated the susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood,spleen,and lymph node specimens,while the nonstructural protein NS1 can activate B cells. Because of the secreted nature of NS1,we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential impacts on systemic immune responses. View Publication

BACKGROUND The Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3,which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered,unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man. METHODS We have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons. RESULTS AZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs,reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO,which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs,reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo,strongly modulated Treg effector molecules (eg,ICOS,CTLA-4,CD25 and 4-1BB),and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy. CONCLUSIONS Antisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669). View Publication

Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells that recognize specific sialic acid modifications on cell surface glycans. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered immune responses and increased tumor growth. In this study,we examined the role of ST8Sia6 on immune cells in regulating antitumor immunity. ST8Sia6 knockout mice had an enhanced immune response to tumors. The loss of ST8Sia6 promoted an enhanced intratumoral activation of macrophages and dendritic cells,including upregulation of CD40. Intratumoral regulatory T cells exhibited a more inflammatory phenotype in ST8Sia6 knockout mice. Using adoptive transfer studies,the change in regulatory T cell phenotype was not cell intrinsic and depended on the loss of ST8Sia6 expression in APCs. Thus,ST8Sia6 generates ligands for Siglecs that dampen antitumor immunity. View Publication