Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth.

Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy in vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.

Generation of B-cell-based cellular vaccine for cancer in murine models.

A B-cell-based cellular vaccine (BVax), produced from 4-1BBL+ B cells, can select tumor-specific B cells that, upon ex vivo culture, can generate tumor-specific antibodies and activate T cells. Here, we present a protocol to generate a B-cell-based vaccine in a CT2A orthotopic glioma murine model. We describe steps for BVax production involving glioma cell implantation, tissue harvesting, 4-1BBL+ B cell isolation, and activation. We also describe assessing BVax phenotype in vitro and in vivo functional status. For complete details on the use and execution of this protocol, please refer to Lee-Chang et al. (2021).1.

B-cells Drive Response to PD-1 Blockade in Glioblastoma Upon Neutralization of TGFß-mediated Immunosuppression.

Immunotherapy has revolutionized cancer treatment but has yet to be translated into brain tumors. Studies in other solid tumors suggest a central role of B-cell immunity in driving immune-checkpoint-blockade efficacy. Using single-cell and single-nuclei transcriptomics of human glioblastoma and melanoma brain metastasis, we found that tumor-associated B-cells have high expression of checkpoint molecules, known to block B-cell-receptor downstream effector function such as plasmablast differentiation and antigen-presentation. We also identified TGFß-1/TGFß receptor-2 interaction as a crucial modulator of B-cell suppression. Treatment of glioblastoma patients with pembrolizumab induced expression of B-cell checkpoint molecules and TGFß-receptor-2. Abrogation of TGFß using different conditional knockouts expanded germinal-center-like intratumoral B-cells, enhancing immune-checkpoint-blockade efficacy. Finally, blocking αVß8 integrin (which controls the release of active TGFß) and PD-1 significantly increased B-cell-dependent animal survival and immunological memory. Our study highlights the importance of intratumoral B-cell immunity and a remodeled approach to boost the effects of immunotherapy against brain tumors.

Antigen-presenting B cells promote TCF-1+ PD1- stem-like CD8+ T-cell proliferation in glioblastoma.

Understanding the spatial relationship and functional interaction of immune cells in glioblastoma (GBM) is critical for developing new therapeutics that overcome the highly immunosuppressive tumor microenvironment. Our study showed that B and T cells form clusters within the GBM microenvironment within a 15-µm radius, suggesting that B and T cells could form immune synapses within the GBM. However, GBM-infiltrating B cells suppress the activation of CD8+ T cells. To overcome this immunosuppression, we leveraged B-cell functions by activating them with CD40 agonism, IFNγ, and BAFF to generate a potent antigen-presenting B cells named BVax. BVax had improved antigen cross-presentation potential compared to naïve B cells and were primed to use the IL15-IL15Ra mechanism to enhance T cell activation. Compared to naïve B cells, BVax could improve CD8 T cell activation and proliferation. Compared to dendritic cells (DCs), which are the current gold standard professional antigen-presenting cell, BVax promoted highly proliferative T cells in-vitro that had a stem-like memory T cell phenotype characterized by CD62L+CD44- expression, high TCF-1 expression, and low PD-1 and granzyme B expression. Adoptive transfer of BVax-activated CD8+ T cells into tumor-bearing brains led to T cell reactivation with higher TCF-1 expression and elevated granzyme B production compared to DC-activated CD8+ T cells. Adoptive transfer of BVax into an irradiated immunocompetent tumor-bearing host promoted more CD8+ T cell proliferation than adoptive transfer of DCs. Moreover, highly proliferative CD8+ T cells in the BVax group had less PD-1 expression than those highly proliferative CD8+ T cells in the DC group. The findings of this study suggest that BVax and DC could generate distinctive CD8+ T cells, which potentially serve multiple purposes in cellular vaccine development.

The CXCL16-CXCR6 axis in glioblastoma modulates T-cell activity in a spatiotemporal context.

Introduction: Glioblastoma multiforme (GBM) pathobiology is characterized by its significant induction of immunosuppression within the tumor microenvironment, predominantly mediated by immunosuppressive tumor-associated myeloid cells (TAMCs). Myeloid cells play a pivotal role in shaping the GBM microenvironment and influencing immune responses, with direct interactions with effector immune cells critically impacting these processes. Methods: Our study investigates the role of the CXCR6/CXCL16 axis in T-cell myeloid interactions within GBM tissues. We examined the surface expression of CXCL16, revealing its limitation to TAMCs, while microglia release CXCL16 as a cytokine. The study explores how these distinct expression patterns affect T-cell engagement, focusing on the consequences for T-cell function within the tumor environment. Additionally, we assessed the significance of CXCR6 expression in T-cell activation and the initial migration to tumor tissues. Results: Our data demonstrates that CXCL16 surface expression on TAMCs results in predominant T-cell engagement with these cells, leading to impaired T-cell function within the tumor environment. Conversely, our findings highlight the essential role of CXCR6 expression in facilitating T-cell activation and initial migration to tumor tissues. The CXCL16-CXCR6 axis exhibits dualistic characteristics, facilitating the early stages of the T-cell immune response and promoting T-cell infiltration into tumors. However, once inside the tumor, this axis contributes to immunosuppression. Discussion: The dual nature of the CXCL16-CXCR6 axis underscores its potential as a therapeutic target in GBM. However, our results emphasize the importance of carefully considering the timing and context of intervention. While targeting this axis holds promise in combating GBM, the complex interplay between TAMCs, microglia, and T cells suggests that intervention strategies need to be tailored to optimize the balance between promoting antitumor immunity and preventing immunosuppression within the dynamic tumor microenvironment.

Laryngeal Manifestations of Ehlers-Danlos Syndrome.

OBJECTIVES: The identification of voice and airway manifestations of Ehlers-Danlos Syndrome (EDS), diagnoses, and potential treatment modalities. STUDY DESIGN: Single institution retrospective case series. METHODS: We examined all patients presenting to our institution over a span of 10 years with a history of EDS or who were subsequently diagnosed with EDS after their evaluation. Demographic and clinical data were collected. RESULTS: Four patients were identified with an underlying diagnosis of EDS. All four patients were heavy voice users. All four patients had history and/or stroboscopy findings suggesting vocal hyperfunction, which we suspect is due to EDS-related hypermobility of the cricoarytenoid joint or fragility of the superficial lamina propria. Two patients also had respiratory symptoms - one with respiratory muscle weakness and sensation loss and one with inducible laryngeal obstruction. All patients were treated with voice therapy with subsequent improvement in their symptoms. CONCLUSIONS: Patients with EDS may present to laryngology clinics with symptoms of dysphonia or dyspnea secondary to their underlying condition. Voice therapy is a low-risk and potentially beneficial treatment in this patient population.

Redox-responsive polyurethane-polyurea nanoparticles targeting to aortic endothelium and atherosclerosis.

Aortic endothelial cell dysfunction is an early trigger of atherosclerosis, the major cause of the cardiovascular disease (CVD). Nanomedicines targeting vascular endothelium and lesions hold great promise as therapeutic solutions to vascular disorders. This study investigates the vascular delivery efficacy of polyurethane-polyurea nanocapsules (Puua-NCs) with pH-synchronized shell cationization and redox-triggered release. Fluorescent lipophilic dye DiI was encapsulated into Puua-NCs of variable sizes and concentrations. In vitro cellular uptake studies with human aortic endothelial cells showed that these Puua-NCs were taken up by cells in a dose-dependent manner. In apolipoprotein E-deficient mice fed a Western diet, a model of atherosclerosis, circulating Puua-NCs were stable and accumulated in aortic endothelium and lesions within 24 hours after intravenous administration. Treatment with thiol-reducing and oxidizing reagents disrupted the disulfide bonds on the surface of internalized NCs, triggering disassembly and intracellular cargo release. Ultimately, Puua-NCs are a potential redox-controllable cardiovascular drug delivery system.

Off-the-Shelf Prostate Stem Cell Antigen-Directed Chimeric Antigen Receptor Natural Killer Cell Therapy to Treat Pancreatic Cancer.

BACKGROUND & AIMS: Pancreatic cancer (PC) is the third leading cause of cancer-related death with a 5-year survival rate of approximately 10%. It typically presents as a late-stage incurable cancer and chemotherapy provides modest benefit. Here, we demonstrate the feasibility, safety, and potency of a novel human natural killer (NK) cell-based immunotherapy to treat PC. METHODS: The expression of prostate stem cell antigen (PSCA) was evaluated in primary PC at messenger RNA and protein levels. The processes of retroviral transduction, expansion, activation, and cryopreservation of primary human NK cells obtained from umbilical cord blood were optimized, allowing us to develop frozen, off-the-shelf, allogeneic PSCA chimeric antigen receptor (CAR) NK cells. The safety and efficacy of PSCA CAR NK cells also expressing soluble (s) interleukin 15 (PSCA CAR_s15 NK cells) were evaluated in vitro and in vivo. RESULTS: PSCA was elevated in primary human PC compared with the adjacent or other normal tissues. PSCA CAR_s15 NK cells displayed significant tumor-suppressive effects against PSCA(+) PC in vitro before and after 1 cycle of freeze-thaw. The viability of frozen PSCA CAR_s15 NK cells persisted more than 90 days in vivo after their last infusion and significantly prolonged the survival of mice engrafted with human PC. CONCLUSIONS: PSCA CAR_s15 NK cells showed therapeutic efficacy in human metastatic PC models without signs of systematic toxicity, providing a strong rationale to support clinical development.

The Interplay between Glioblastoma and Its Microenvironment.

GBM is the most common primary brain tumor in adults, and the aggressive nature of this tumor contributes to its extremely poor prognosis. Over the years, the heterogeneous and adaptive nature of GBM has been highlighted as a major contributor to the poor efficacy of many treatments including various immunotherapies. The major challenge lies in understanding and manipulating the complex interplay among the different components within the tumor microenvironment (TME). This interplay varies not only by the type of cells interacting but also by their spatial distribution with the TME. This review highlights the various immune and non-immune components of the tumor microenvironment and their consequences f the efficacy of immunotherapies. Understanding the independent and interdependent aspects of the various sub-populations encapsulated by the immune and non-immune components will allow for more targeted therapies. Meanwhile, understanding how the TME creates and responds to different environmental pressures such as hypoxia may allow for other multimodal approaches in the treatment of GBM. Ultimately, a better understanding of the GBM TME will aid in the development and advancement of more effective treatments and in improving patient outcomes.

Current Immunotherapeutic Strategies for the Treatment of Glioblastoma.

Glioblastoma (GBM) is a lethal primary brain tumor. Despite extensive effort in basic, translational, and clinical research, the treatment outcomes for patients with GBM are virtually unchanged over the past 15 years. GBM is one of the most immunologically "cold" tumors, in which cytotoxic T-cell infiltration is minimal, and myeloid infiltration predominates. This is due to the profound immunosuppressive nature of GBM, a tumor microenvironment that is metabolically challenging for immune cells, and the low mutational burden of GBMs. Together, these GBM characteristics contribute to the poor results obtained from immunotherapy. However, as indicated by an ongoing and expanding number of clinical trials, and despite the mostly disappointing results to date, immunotherapy remains a conceptually attractive approach for treating GBM. Checkpoint inhibitors, various vaccination strategies, and CAR T-cell therapy serve as some of the most investigated immunotherapeutic strategies. This review article aims to provide a general overview of the current state of glioblastoma immunotherapy. Information was compiled through a literature search conducted on PubMed and clinical trials between 1961 to 2021.

Polyamines drive myeloid cell survival by buffering intracellular pH to promote immunosuppression in glioblastoma.

Glioblastoma is characterized by the robust infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs). It is not fully understood how TAMCs survive in the acidic tumor microenvironment to cause immunosuppression in glioblastoma. Metabolic and RNA-seq analysis of TAMCs revealed that the arginine-ornithine-polyamine axis is up-regulated in glioblastoma TAMCs but not in tumor-infiltrating CD8+ T cells. Active de novo synthesis of highly basic polyamines within TAMCs efficiently buffered low intracellular pH to support the survival of these immunosuppressive cells in the harsh acidic environment of solid tumors. Administration of difluoromethylornithine (DFMO), a clinically approved inhibitor of polyamine generation, enhanced animal survival in immunocompetent mice by causing a tumor-specific reduction of polyamines and decreased intracellular pH in TAMCs. DFMO combination with immunotherapy or radiotherapy further enhanced animal survival. These findings indicate that polyamines are used by glioblastoma TAMCs to maintain normal intracellular pH and cell survival and thus promote immunosuppression during tumor evolution.

Activation of 4-1BBL+ B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma.

Immunotherapy has revolutionized the treatment of many tumors. However, most glioblastoma (GBM) patients have not, so far, benefited from such successes. With the goal of exploring ways to boost anti-GBM immunity, we developed a B cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism and IFNγ stimulation. BVax migrates to key secondary lymphoid organs and is proficient at antigen cross-presentation, which promotes both the survival and the functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. This treatment elicited immunological memory that prevented the growth of new tumors upon subsequent reinjection in cured mice. GBM patient-derived BVax was successful in activating autologous CD8+ T cells; these T cells showed a strong ability to kill autologous glioma cells. Our study provides an efficient alternative to current immunotherapeutic approaches that can be readily translated to the clinic.

A Notch3-Marked Subpopulation of Vascular Smooth Muscle Cells Is the Cell of Origin for Occlusive Pulmonary Vascular Lesions.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by profound vascular remodeling in which pulmonary arteries narrow because of medial thickening and occlusion by neointimal lesions, resulting in elevated pulmonary vascular resistance and right heart failure. Therapies targeting the neointima would represent a significant advance in PAH treatment; however, our understanding of the cellular events driving neointima formation, and the molecular pathways that control them, remains limited. METHODS: We comprehensively map the stepwise remodeling of pulmonary arteries in a robust, chronic inflammatory mouse model of pulmonary hypertension. This model demonstrates pathological features of the human disease, including increased right ventricular pressures, medial thickening, neointimal lesion formation, elastin breakdown, increased anastomosis within the bronchial circulation, and perivascular inflammation. Using genetic lineage tracing, clonal analysis, multiplexed in situ hybridization, immunostaining, deep confocal imaging, and staged pharmacological inhibition, we define the cell behaviors underlying each stage of vascular remodeling and identify a pathway required for neointima formation. RESULTS: Neointima arises from smooth muscle cells (SMCs) and not endothelium. Medial SMCs proliferate broadly to thicken the media, after which a small number of SMCs are selected to establish the neointima. These neointimal founder cells subsequently undergoing massive clonal expansion to form occlusive neointimal lesions. The normal pulmonary artery SMC population is heterogeneous, and we identify a Notch3-marked minority subset of SMCs as the major neointimal cell of origin. Notch signaling is specifically required for the selection of neointimal founder cells, and Notch inhibition significantly improves pulmonary artery pressure in animals with pulmonary hypertension. CONCLUSIONS: This work describes the first nongenetically driven murine model of pulmonary hypertension (PH) that generates robust and diffuse occlusive neointimal lesions across the pulmonary vascular bed and does so in a stereotyped timeframe. We uncover distinct cellular and molecular mechanisms underlying medial thickening and neointima formation and highlight novel transcriptional, behavioral, and pathogenic heterogeneity within pulmonary artery SMCs. In this model, inflammation is sufficient to generate characteristic vascular pathologies and physiological measures of human PAH. We hope that identifying the molecular cues regulating each stage of vascular remodeling will open new avenues for therapeutic advancements in the treatment of PAH.

Myeloid-Derived Suppressive Cells Promote B cell-Mediated Immunosuppression via Transfer of PD-L1 in Glioblastoma.

The potent immunosuppression induced by glioblastoma (GBM) is one of the primary obstacles to finding effective immunotherapies. One hallmark of the GBM-associated immunosuppressive landscape is the massive infiltration of myeloid-derived suppressor cells (MDSC) and, to a lesser extent, regulatory T cells (Treg) within the tumor microenvironment. Here, we showed that regulatory B cells (Breg) are a prominent feature of the GBM microenvironment in both preclinical models and clinical samples. Forty percent of GBM patients (n = 60) scored positive for B-cell tumor infiltration. Human and mouse GBM-associated Bregs were characterized by immunosuppressive activity toward activated CD8+ T cells, the overexpression of inhibitory molecules PD-L1 and CD155, and production of immunosuppressive cytokines TGFß and IL10. Local delivery of B cell-depleting anti-CD20 immunotherapy improved overall survival of animals (IgG vs. anti-CD20 mean survival: 18.5 vs. 33 days, P = 0.0001), suggesting a potential role of Bregs in GBM progression. We unveiled that GBM-associated MDSCs promoted regulatory B-cell function by delivering microvesicles transporting membrane-bound PD-L1, able to be up-taken by tumoral B cells. The transfer of functional PD-L1 via microvesicles conferred Bregs the potential to suppress CD8+ T-cell activation and acquisition of an effector phenotype. This work uncovered the role of B cells in GBM physiopathology and provides a mechanism by which the GBM microenvironment controls B cell-mediated immunosuppression.See related Spotlight on p. 1902.

Forming a support group for people affected by inflammatory bowel disease.

Inflammatory bowel disease (IBD) - primarily Crohn's disease and ulcerative colitis - is a debilitating lifelong condition with significant health and economic costs. From diagnosis to management, IBD can cause huge psychosocial concerns to patients and their caregivers. This study reports an experience of a Crohn's patient, leading to the formation of the first IBD patient support group in Singapore and how this group has evolved in the last 4 years in supporting other IBD patients. IBD patient advocacy and/or support groups facilitate open conversations on patients' fears, concerns, preferences and needs, and may potentially improve disease knowledge and quality of life for individuals with the condition or their families.

Initial respiratory support with cold, dry gas versus heated humidified gas and admission temperature of preterm infants.

OBJECTIVE: To assess whether the addition of heated humidified gas (HHG) at delivery and until neonatal unit arrival improved admission temperatures of preterm infants. STUDY DESIGN: This multicenter, randomized controlled trial was performed in New Zealand and The Netherlands. Infants <32 weeks' gestation who required respiratory support after delivery were randomized to either cold, dry gas or HHG from birth. Standard measures to prevent hypothermia included heated delivery rooms, the use of radiant warmers, body wrap, and head covering. The primary outcome was axillary temperature in the normothermic (36.5-37.5°C) range on admission to a neonatal intensive care unit. Secondary outcomes were measures of respiratory support and neonatal morbidities. The effect of humidification was analyzed by the use of logistic regression. RESULTS: Of 203 randomized infants, 100 received HHG (humidifier set to 37°C) and 103 received cold, dry gas. In the HHG group, 69 (69%) were normothermic compared with 57 (55%) in the cold, dry gas group (unadjusted OR 1.8, 95% CI 1.01-3.19). A greater number of infants <28 weeks were normothermic on admission in the HHG group (24/35; ie, 69%) compared with the cold, dry gas group (16/38; ie, 42%; P = .03). In addition, 2 (2%) infants in the HHG group had admission temperatures <35.5°C compared with 12 (12%) in the cold, dry gas group (P = .007). Respiratory and short-term outcomes were not different. CONCLUSION: Adding HHG during respiratory support in preterm infants from birth increased the incidence of normothermia at admission.

Microneedle electrodes toward an amperometric glucose-sensing smart patch.

Here, efforts toward the development of a microneedle-based glucose sensor or "smart patch" for intradermal glucose sensing are described. Metallic microneedle array electrodes, conducting polymers, and glucose oxidase form the sensor platform. This work represents the first steps toward the development of painless, transdermal-sensing devices for continuous glucose monitoring.

Cerebellar haemorrhage in the extremely preterm infant.

AIM: The aim of this study was to investigate the incidence, risk factors and developmental outcomes of cerebellar haemorrhage in the extremely preterm infant. METHODS: Over a 4-year period from January 2004, all patients with cerebellar haemorrhage born at 24-27 weeks gestation or with a birthweight < 1000 g were identified from database review. All patients had cranial ultrasound scans including views from the mastoid fontanelle. To verify the incidence, a review of all reports over the 4-year period and a review of all cranial imaging over a 2-year period were carried out. RESULTS: From the data analysed on 339 neonates, five cases of cerebellar haemorrhage were identified, four on ultrasound scan (1.2%) and one on magnetic resonance imaging (1.5% total). Two cases were associated with grade III peri-intraventricular haemorrhage. The cases had diverse maternal, antenatal and post-natal risk factors. The two with associated peri-intraventricular haemorrhage have developmental delay. The cases with isolated cerebellar haemorrhage had good neurodevelopmental outcomes. CONCLUSIONS: A low incidence of cerebellar haemorrhage identified from the mastoid fontanelle was demonstrated. The neurodevelopmental outcome was better than that described in previous reports.

Heating of gases during neonatal resuscitation: a bench study.

AIM: Standard practice within the neonatal unit is to use heated humidified gas as it decreases respiratory complications in neonates requiring respiratory support. Using cold unhumidified gases during resuscitation could potentially cool the baby as well as exacerbate potential lung injury. We aimed to study the temperature and humidity aspects of using heated, humidified gas for neonatal resuscitation. METHODS: A heated patient circuit was connected to a T-piece resuscitator via a humidifier. An oxygen flowmeter was set at 10 L/min. Temperature recordings at the humidifier chamber (T1), distal temperature probe (T2) and T-piece (T3) were taken over 20 min at 30s intervals. A humidity sensor was placed at T3. RESULTS: Target temperatures were not reached. Time to 36°C (mean (sd)): T1 11.1 min (1.71); T3 11.6 min (1.77). T2 took 13.6 min (1.07) to reach 39°C. T1 and T3 were within ±1°C at 5.1 min (0.6). A biphasic relationship demonstrated the time lag between the temperatures of the heated patient circuit and the humidifier chamber. T3 strongly correlated to T1 when T1 is ≥28°C (r(2)=0.85). Humidity was difficult to measure and results were inferred from temperature recordings. CONCLUSION: This in vitro test showed that heated, humidified gas is possible during neonatal resuscitation. Adequate time must be allowed for the humidifier chamber to warm to near optimal temperature. The patient circuit is initially heated faster than the humidifier chamber. The displayed T1 temperature correlates to the temperature at T3 at ≥28°C.

Non-alcoholic steatohepatitis mimicking liver metastasis in obesity surgery.

We describe a case of non-alcoholic steatohepatitis detected incidentally during laparoscopic Roux-en-Y gastric bypass (LRYGBP). A 51-year-old female patient was scheduled for elective LRYGBP. Her weight was 144.9 kg and her body mass index was 56. Liver function tests showed mild elevation in alanine transaminase. The patient had a history of hypertension and insulin resistance and had no history of alcohol abuse. During LRYGBP, the gross appearance of the liver resembled metastatic lesions but the histology confirmed the diagnosis of non-alcoholic steatohepatitis. The appearance of the liver necessitated taking biopsies, which showed Von Meyenburg complexes and moderate macrovesicular steatosis. This patient made an uneventful recovery and was discharged 2 days postoperatively.