Gregor Hutter

PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin’s lymphoma. Although it is well established that PD-1–PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1–PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1–PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.

No Superoxide Dismutase Activity of Cellular Prion Protein in vivo

Abstract Prion diseases are characterized by the deposition of PrPSc, an abnormal form of the cellular prion protein PrPc, which is encoded by the Prnp gene. PrPc is highly expressed on neurons and its function is unknown. Recombinant PrPc was claimed to possess superoxide dismutase (SOD) activity, and it was hypothesized that abrogation of this function may contribute to neurodegeneration in prion diseases. We tested this hypothesis in vivo by studying copper/zinc and manganese SOD activity in genetically defined crosses of mice lacking the Sod1 gene with mice lacking PrPc, and with hemizygous or homozygous tga20 transgenic mice overexpressing various levels of PrPc. We failed to detect any influence of the Prnp genotype and gene dosage on SOD1 or SOD2 activity in heart, spleen, brain, and synaptosomeenriched brain fractions. Control experiments included crosses of mice lacking or overexpressing PrP with mice overexpressing human Cu[2+]/Zn[2+]-superoxide dismutase, and confirmed that SOD enzymatic activity correlated exclusively with the gene dosage of bona fide human or murine SOD. We conclude that PrPc in vivo does not discernibly contribute to total SOD activity and does not possess an intrinsic dismutase activity.

IDH/MGMT-driven molecular classification of low-grade glioma is a strong predictor for long-term survival

BACKGROUND Low-grade gliomas (LGGs) are rare brain neoplasms, with survival spanning up to a few decades. Thus, accurate evaluations on how biomarkers impact survival among patients with LGG require long-term studies on samples prospectively collected over a long period. METHODS The 210 adult LGGs collected in our databank were screened for IDH1 and IDH2 mutations (IDHmut), MGMT gene promoter methylation (MGMTmet), 1p/19q loss of heterozygosity (1p19qloh), and nuclear TP53 immunopositivity (TP53pos). Multivariate survival analyses with multiple imputation of missing data were performed using either histopathology or molecular markers. Both models were compared using Akaikes information criterion (AIC). The molecular model was reduced by stepwise model selection to filter out the most critical predictors. A third model was generated to assess for various marker combinations. RESULTS Molecular parameters were better survival predictors than histology (ΔAIC = 12.5, P< .001). Forty-five percent of studied patients died. MGMTmet was positively associated with IDHmut (P< .001). In the molecular model with marker combinations, IDHmut/MGMTmet combined status had a favorable impact on overall survival, compared with IDHwt (hazard ratio [HR] = 0.33, P< .01), and even more so the triple combination, IDHmut/MGMTmet/1p19qloh (HR = 0.18, P< .001). Furthermore, IDHmut/MGMTmet/TP53pos triple combination was a significant risk factor for malignant transformation (HR = 2.75, P< .05). CONCLUSION By integrating networks of activated molecular glioma pathways, the model based on genotype better predicts prognosis than histology and, therefore, provides a more reliable tool for standardizing future treatment strategies.

The role of calorie restriction and SIRT1 in prion-mediated neurodegeneration

A central focus of aging research is to determine how calorie restriction (CR) extends lifespan and delays diseases of aging. SIRT1, the mammalian ortholog of Sir2 in yeast, is a longevity factor which mediates dietary restriction in diverse species. In addition, SIRT1 plays a protective role in several models of neurodegenerative disease. We tested the role of SIRT1 in mediating the effects of CR in a mouse model of prion disease. Prion diseases are protein misfolding disorders of the central nervous system with many similarities to other neurodegenerative diseases, including deposition of aggregated protein, gliosis, and loss of synapses and neurons. We report that the onset of prion disease is delayed by CR and in the SIRT1 KO mice fed ad libitum. CR exerts no further effect on the SIRT1 KO strain, suggesting the effects of CR and SIRT1 deletion are mechanistically coupled. In conjunction, SIRT1 is downregulated in certain brain regions of CR mice. The expression of PrP mRNA and protein is reduced in the brains of CR mice and in SIRT1 knockout mice, suggesting a possible mechanism for the delayed onset of disease, as PrP levels are a critical determinant of how quickly mice succumb to prion disease. Surprisingly, CR greatly shortens the duration of clinical symptoms of prion disease and ultimately shortens lifespan of prion-inoculated mice in a manner that is independent of SIRT1. Taken together, our results suggest a more complex interplay between CR, SIRT1, and neurodegenerative diseases than previously appreciated.

Heat shock factor 1 regulates lifespan as distinct from disease onset in prion disease

Prion diseases are fatal, transmissible, neurodegenerative diseases caused by the misfolding of the prion protein (PrP). At present, the molecular pathways underlying prion-mediated neurotoxicity are largely unknown. We hypothesized that the transcriptional regulator of the stress response, heat shock factor 1 (HSF1), would play an important role in prion disease. Uninoculated HSF1 knockout (KO) mice used in our study do not show signs of neurodegeneration as assessed by survival, motor performance, or histopathology. When inoculated with Rocky Mountain Laboratory (RML) prions HSF1 KO mice had a dramatically shortened lifespan, succumbing to disease ≈20% faster than controls. Surprisingly, both the onset of home-cage behavioral symptoms and pathological alterations occurred at a similar time in HSF1 KO and control mice. The accumulation of proteinase K (PK)-resistant PrP also occurred with similar kinetics and prion infectivity accrued at an equal or slower rate. Thus, HSF1 provides an important protective function that is specifically manifest after the onset of behavioral symptoms of prion disease.

SIRPα polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells

The regulation of phagocytosis previously ascribed to prion protein PrPC is found to be controlled by the linked locus encoding SIRPα.

Disrupting the CD47-SIRPα anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors

Anti-CD47 antibody is effective for treating malignant pediatric brain tumors without detectable toxicity in patient-derived xenograft models. Brain tumors, meet macrophages A protein called CD47 is often expressed on the surface of tumor cells, where it serves as a “don’t eat me” signal that blocks macrophages from attacking the tumor. To overcome this signal and allow the macrophages to “eat” tumor cells, Gholamin et al. engineered a humanized antibody that blocks CD47 signaling. The researchers tested the efficacy of this antibody in patient-derived xenograft models of a variety of pediatric brain tumors. The treatment was successful at inhibiting CD47, killing tumor cells, and prolonging the animals’ survival, all without toxic effects on normal tissues. Morbidity and mortality associated with pediatric malignant primary brain tumors remain high in the absence of effective therapies. Macrophage-mediated phagocytosis of tumor cells via blockade of the anti-phagocytic CD47-SIRPα interaction using anti-CD47 antibodies has shown promise in preclinical xenografts of various human malignancies. We demonstrate the effect of a humanized anti-CD47 antibody, Hu5F9-G4, on five aggressive and etiologically distinct pediatric brain tumors: group 3 medulloblastoma (primary and metastatic), atypical teratoid rhabdoid tumor, primitive neuroectodermal tumor, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Hu5F9-G4 demonstrated therapeutic efficacy in vitro and in vivo in patient-derived orthotopic xenograft models. Intraventricular administration of Hu5F9-G4 further enhanced its activity against disseminated medulloblastoma leptomeningeal disease. Notably, Hu5F9-G4 showed minimal activity against normal human neural cells in vitro and in vivo, a phenomenon reiterated in an immunocompetent allograft glioma model. Thus, Hu5F9-G4 is a potentially safe and effective therapeutic agent for managing multiple pediatric central nervous system malignancies.

Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.

Abdominal seeding of an atypical teratoid/rhabdoid tumor of the pineal gland along a ventriculoperitoneal shunt catheter

Atypical teratoid/rhabdoid tumor (AT/RT) is a rather novel malignant central nervous system (CNS) neoplasm of uncertain origin, which was first defined as an entity in 1987 [5]. The vast majority of AT/RTs occur in infants or children below 5 years of age and only few AT/ RTs in adults have been reported [3, 6]. Most AT/RTs arise in the posterior fossa, the cerebral hemispheres and the suprasellar region, but can also occur in the spinal cord and the pineal region. In the latter, AT/RT has been reported only once [10]. Histopathologically, AT/ RT is composed of rhabdoid cells with or without areas of epithelial, mesenchymal and/or primitive neuroectodermal differentiation. Fluorescence in situ hybridization (FISH) and loss of heterozygosity studies revealed deletions of chromosome 22 in 75–90% of AT/RTs with inactivation of the INI1/hSNF5 gene and, consequently, lack of INI1 protein expression similar to rhabdoid tumors of other organs such as kidney [1, 6]. Despite the aggressive treatment with surgery, chemoand radiotherapy, AT/RTs have an unfavorable prognosis with early recurrence and short median survival times [8]. An otherwise healthy 45-year-old-woman presented with visual disturbances. Cranial magnetic resonance imaging (MRI) displayed a tumor mass in the pineal region, which was resected. Histological examination revealed a neoplasm with high cellularity, numerous mitotic figures and a predominantly rhabdoid appearance of the tumor cells (Fig. 1a, b). Immunohistochemical analysis revealed strong reactivity for vimentin in almost all, and focal positivity for smooth muscle actin (SMA), cytokeratins and epithelial membrane antigen (EMA) in some tumor cells (Fig. 1c–f). FISH analysis using probes to the breakpoint cluster region (BCR) on chromosome 22q11.2 showed a single BCR signal in tumor cells, while two signals of the ABL locus on chromosome 9 were present. This result suggested deletion of the INI1/hSNF5 gene, which is located close to the BCR locus on chromosome 22q11.2 (Fig. 1h). This conclusion was supported by the loss of INI1/ hSNF5 protein expression in tumor cells, whereas nonneoplastic CNS cells displayed a nuclear INI1 signal (Fig. 1g). Histological, immunohistochemical and FISH results were consistent with the diagnosis of AT/RT, which was confirmed by the Brain Tumor Reference Center of the German Society of Neuropathology and Neuroanatomy. Postoperative radioand chemotherapy was performed. Due to development of occlusive hydrocephalus, a ventriculoperitoneal (VP) shunt catheter was inserted. Six months after the initial diagnosis, the tumor recurred locally. Gross total resection was achieved and histological and immunohistochemical analysis re-confirmed the initial diagnosis of an AT/RT. One month after re-craniotomy, the patient was admitted to the hospital with progressive loss of B. Ingold Æ W. Jochum Institute for Surgical Pathology, University Hospital Zurich, 8091 Zurich, Switzerland E-mail: wolfram.jochum@usz.ch Tel.: +41-44-2552512 Fax: +41-44-2554416

Risk factors for postoperative CSF leakage after elective craniotomy and the efficacy of fleece-bound tissue sealing against dural suturing alone: a randomized controlled trial.

OBJECT Cerebrospinal fluid leakage is an immanent risk of cranial surgery with dural opening. Recognizing the risk factors for this complication and improving the technique of dural closure may reduce the associated morbidity and its surgical burden. The aim of this paper was to investigate whether the addition of TachoSil on top of the dural suture reduces postoperative CSF leakage compared with dural suturing alone and to assess the frequency and risk factors for dural leakage and potentially related complications after elective craniotomy. METHODS The authors conducted a prospective, randomized, double-blinded single-center trial in patients undergoing elective craniotomy with dural opening. They compared their standard dural closure by running suture alone (with the use of a dural patch if needed) to the same closure with the addition of TachoSil on top of the suture. The primary end point was the incidence of CSF leakage, defined as CSF collection or any open CSF fistula within 30 days. Secondary end points were the incidence of infection, surgical revision, and length of stay in the intensive care unit (ICU) or intermediate care (IMC) unit. The site of craniotomy, a history of diabetes mellitus, a diagnosis of meningioma, the intraoperative need of a suturable dural substitute, and blood parameters were assessed as potential risk factors for CSF leakage. RESULTS The authors enrolled 241 patients, of whom 229 were included in the analysis. Cerebrospinal fluid leakage, mostly self-limiting subgaleal collections, occurred in 13.5% of patients. Invasive treatment was performed in 8 patients (3.5%) (subgaleal puncture in 6, lumbar drainage in 1, and surgical revision in 1 patient). Diabetes mellitus, a higher preoperative level of C-reactive protein (CRP), and the intraoperative need for a dural patch were positively associated with the occurrence of the primary end point (p = 0.014, 0.01, and 0.049, respectively). Cerebrospinal fluid leakage (9.7% vs 17.2%, OR 0.53 [95% CI 0.23-1.15], p = 0.108) and infection (OR 0.18 [95% CI 0.01-1.18], p = 0.077) occurred less frequently in the study group than in the control group. TachoSil significantly reduced the probability of staying in the IMC unit for 1 day or longer (OR 0.53 [95% CI 0.27-0.99], p = 0.048). Postoperative epidural hematoma and empyema occurred in the control group but not in the study group. CONCLUSIONS Dural leakage after elective craniotomy/durotomy occurs more frequently in association with diabetes mellitus, elevated preoperative CRP levels, and the intraoperative need of a dural patch. This randomized controlled trial showed no statistically significant reduction of postoperative CSF leakage and surgical site infections upon addition of TachoSil on the dural suture, but there was a significant reduction in the length of stay in the IMC unit. Dural augmentation with TachoSil was safe and not related to adverse events. Clinical trial registration no. NCT00999999 ( http://www.ClinicalTrials.gov ).