Biology subjects

Woulfe, J.

Publications and source records attributed to Woulfe, J..

4 recordsLinked to original sources

bioRxiv2026

Characterization of tumor interactions with the immune system in an autochthonous mouse model of glioblastoma

BackgroundGlioblastoma is an aggressive and incurable brain tumor. Clinical trials of immune checkpoint inhibitors showed no clinical benefit in glioblastoma when given after surgery. However, a clinical trial in which PD1 inhibition was given prior to second surgery did show pharmacodynamic evidence for activity. This suggests the possibility that immune checkpoint inhibitors may be more effective in a setting where large tumors are present. Here we have studied immune responses to large tumors in an autochthonous mouse model of glioblastoma. MethodsGlioblastoma was induced by transfection with oncogenic plasmids injected directly into the lateral ventricle of neonatal mice. Immune responses were assessed using a combination of spectral flow cytometry and immunohistochemistry. ResultsThere was a marked immune response to large tumors, with significant increases in CD4 T cells and dendritic cells. T cell changes occurred primarily at leptomeningeal/perivascular border sites. A large proportion of CD4 T cells expressed PD1 and half of these were regulatory T cells. NK cells were also increased in mice with large tumors, but were predominantly in immature states. The mouse model accurately recapitulates the formation of palisading necroses. These contain apoptotic cells and avidly recruit myeloid cells that are induced to express large amounts of TGF{beta}. ConclusionsLarge glioblastoma tumors generate a border site population of PD1 positive T cells that may explain the pharmacodynamic response in neoadjuvant trials, and a palisading necrosis-driven immunosuppressive mechanism that may explain why responses are insufficient to provide a significant clinical benefit. KEY POINTSThe SB mouse model accurately recapitulates immune features of human glioblastoma Large tumors induce a significant border site immune response Palisading necroses in large tumors counter this with a strong immunosuppressive response IMPORTANCE OF STUDYImmune checkpoint inhibitors have not shown efficacy in glioblastoma when used post-surgery, but do show pharmacodynamic activity when used in patients prior to second surgery (i.e. neoadjuvant). This suggest the possibility that immune checkpoint inhibition is more effective when large tumors are present. Using a clinically-relevant autochthonous mouse model, we show here that large tumors induce an immune response that is evident in leptomeningeal border sites. Large tumors in this mouse model also generate palisading necroses, a well-known diagnostic feature in glioblastoma tumors. These palisading necroses generate large amounts of TGF{beta}, providing a mechanism by which large tumors can suppress border site immune responses. This further supports the concept that palisading necroses are drivers of glioblastoma malignancy and suggests novel strategies to enhance responses to immune checkpoint inhibition in this cancer.

cancer biology↗

bioRxiv2025

Reinke crystals are immunoreactive for purine-synthesizing metabolic enzymes

Reinke crystals are a defining histological feature of human adult Leydig cells, the testosterone producing cells of the testis. These structures are present in the cytoplasm and the nucleus and display quantitative alterations in a variety of physiological and pathological contexts. The functional significance and protein composition of Reinke crystals have remained elusive for over a century. Here, we demonstrate that Reinke crystals are intensely immunoreactive for inosine monophosphate dehydrogenase (IMPDH), and phosphoribosyl pyrophosphate synthetase (PRPS), two key rate-limiting enzymes in the de novo synthesis of purine nucleotides. IMPDH and PRPS are two of several metabolic enzymes that are capable of forming mesoscale filamentous aggregates as a mechanism to regulate enzyme activity. IMPDH is also able to form crystals in cellulo. Our observations link Reinke crystal formation to purine nucleotide metabolism in Leydig cells. We discuss how this novel finding may relate to the unique dependence of Leydig cells on guanyl-based purine nucleotides for testosterone synthesis. The results of this study may have important implications for understanding metabolic contributions to male reproductive disorders as well as offering a novel diagnostic and theranostic tool applicable to Leydig cell neoplasms.

pathology↗

bioRxiv2024

Transglutaminase 2 function in glioblastoma tumor efferocytosis

Glioblastoma is an aggressive and incurable type of brain cancer. Regions of tissue necrosis are a distinctive pathological feature of this cancer. These arise through thrombosis of tumor vasculature, driven by tumor-derived pro-coagulation factors. In studies of transglutaminase 2 (TGM2), we observed that TGM2 mRNA expression in glioblastoma was primarily in a subset of tumor-infiltrating myeloid cells with hypoxia gene expression signatures. Analysis of xenograft and human glioblastoma samples by immunohistochemistry showed that macrophages in the vicinity of necrotic regions expressed very high levels of TGM2. These macrophages were engaged in the phagocytosis of apoptotic cells, a process known as efferocytosis. In cell culture, incubation of macrophages with apoptotic cells induced TGM2 expression in macrophages, and TGM2 inhibitors blocked efferocytosis. In patient-derived glioblastoma organoids cultured in 5% O2, a basal level of apoptosis was observed, and endogenous macrophages were observed in the process of clearing apoptotic cells. Clearance of apoptotic cells was reduced in organoids treated with a TGM2 inhibitor. Apoptotic cells and efferocytosis were both markedly lower in organoids grown in 20% O2. These data, together with previous work, define a model in which necrotic regions in glioblastoma induce hypoxia-driven apoptosis, which in turn promotes efferocytosis by macrophages. TGM2 is both a marker of efferocytosis and a target for efferocytosis inhibition in this process. Efferocytosis is a potent immunosuppressive mechanism, so this process provides an additional mechanism by which large glioblastoma tumors can evade immune responses.

cancer biology↗

bioRxiv2020

Age-Associated Insolubility of Parkin in Human Midbrain is Linked to Redox Balance and Sequestration of Reactive Dopamine Metabolites

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions, which are reflected in its posttranslational modifications. We found that in human control brain, including the S. nigra, parkin is largely insoluble after age 40 years, which is linked to its oxidation, e.g., at Cys95 and Cys253. In mice, oxidative stress increases posttranslational modifications at parkin cysteines and reduces its solubility. Oxidation of recombinant parkin also promotes insolubility and aggregate formation, but in parallel, lowers hydrogen peroxide (H2O2). This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. Intriguingly, in parkin-deficient human brain H2O2 concentrations are elevated. In prkn-null mice, H2O2 levels are dysregulated under oxidative stress conditions, such as acutely by MPTP-toxin exposure or chronically due to a second genetic hit. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic M17 cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at primate-specific Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation. In sections of normal, adult human midbrain, parkin specifically co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these redox effects may augment oxidative stress in dopamine producing neurons of mutant PRKN allele carriers, thereby contributing to neurodegeneration.

neuroscience↗