Biology subjects

Ebert, L. M.

Publications and source records attributed to Ebert, L. M..

6 recordsLinked to original sources

bioRxiv2026

Tumor Protein D54 (TPD54) regulates intracellular protein trafficking, cellular function and disease progression in melanoma

To facilitate survival, migration and evasion of immune surveillance, cancer cells tightly coordinate the synthesis and trafficking of a diverse repertoire of proteins to their cell surface and the surrounding tumor microenvironment. A key mechanism underlying this process is the intracellular membrane trafficking pathways, including vesicular transport systems. There remains a paucity of mechanistic insight into the regulatory components that mediate nascent protein trafficking and their dysregulation in cancer. Herein, we investigate Tumor Protein D54 (TPD54) as a central regulator of intracellular protein transport that is exploited by melanoma cells to promote disease progression. Integrative analyses of patient-derived tumor tissue specimens show that the expression of TPD52L2 (the gene encoding TPD54) is frequently overexpressed in melanoma and correlates with adverse clinical outcomes, including reduced responses to immune checkpoint blockade. Mechanistic investigations further revealed that TPD54 maintains Golgi integrity and orchestrates trafficking of early endosomes, anterograde vesicles and extracellular vesicles. Functionally, TPD54 augments the secretion of pro-cancerous cytokines, increases the cell surface expression of adhesion-signaling receptors (e.g. integrin-{beta}1 and desmoglein-2), promotes melanoma cell migration and elevates their capability to undergo vasculogenic mimicry. Targeting TPD52L2 in two mouse models of melanoma (B16-F10 and HCmel12) showed significant attenuation of tumor growth, disrupted tumor vasculature, enhanced anti-tumor immunity with infiltration of CD8+ T cells and reduced metastatic disease. Collectively, these findings establish TPD54 as a critical and previously underappreciated regulator of protein trafficking in cancer cells that directly contributes to disease progression and highlights its potential as a novel therapeutic target to combat melanoma.

cancer biology↗

bioRxiv2025

Tumors Located in the Brain Impair the Frequency and Phenotype of Dendritic Cells in Blood and Tumor

BackgroundProfessional antigen-presenting dendritic cells (DC) are critical for anti-tumor immune responses, yet patients with glioblastoma, an aggressive primary brain tumor that responds poorly to current investigational immunotherapies, appear to be deficient in DC. The extent of this deficiency, the specific DC subsets affected, and the causative mechanisms remain undefined. Furthermore, DCs in other brain tumors have not been systematically investigated. MethodsHigh-parameter flow cytometry was used to profile circulating and intra-tumoral DCs in patients with glioblastoma, low-grade gliomas, and brain metastases, and non-CNS cancers. Plasma DC growth factors were quantified using ELISA. We also evaluated single-cell RNA sequencing (scRNAseq) datasets to compare intra-tumoral DCs in brain and lung tumors, and quantified DC number and phenotype in three intracranial mouse brain tumor models. ResultsOur studies reveal a profound systemic reduction of multiple DC subsets in the blood of patients with diverse brain tumors, coupled with reduced DC activation marker expression and lower plasma levels of FLT3L and G-CSF. Furthermore, scRNAseq analyses revealed reduced intra-tumoral DCs in glioblastoma compared to lung tumors. Circulating DC numbers inversely correlated with perioperative corticosteroid dose in patients with or without a brain tumor. However, brain tumor patients not receiving corticosteroids also had reduced DCs, suggesting a direct effect of the brain tumor. This was supported by our observation of systemic DC defects in mouse brain tumor models. ConclusionsWe reveal profound DC defects in patients with brain tumors, which may contribute to current difficulties in developing effective immunotherapies for glioblastoma. SummaryWe demonstrate multiple DC defects in patients with brain tumors. This includes a profound reduction in circulating DC number, diminished activation marker expression and growth factor levels in cancer patients with brain tumors compared to those without, and reduced intra-tumoral DCs in brain compared to lung tumors. This is the first time DC subsets have been fully characterized in a range of brain tumor patients. We show that corticosteroid usage is closely associated with DC defects, highlighting the adverse effects of a standard symptomatic treatment on these critical immune cells. However, tumors located within the brain also directly contribute to DC defects. We identified several mouse brain tumor models that can be used to further the understanding of this endogenous DC deficiency and to develop approaches to restore DCs, ultimately leading to new combination immunotherapies for the treatment of brain cancers. HighlightsO_LIDCs are reduced in brain tumor patients and mice with intracranial tumors. C_LIO_LIDCs are rare within glioblastoma tumor tissue. C_LIO_LIThe presence of a brain tumor and corticosteroid use are both associated with DC defects. C_LI

immunology↗

bioRxiv2025

A Characterization of the Immune Cells in Immunocompetent and Immunodeficient Mice with Orthotopic Brain Tumors.

BackgroundGlioblastoma is characterized by poor survival with few treatment advances for over 20 years. Active research is being conducted in immunotherapies; however, this may be hampered by the lack of detailed characterization of the immune compartments of orthotopic murine brain tumor models. MethodsWe used cell lines derived from glioblastoma patients and murine glioma cell lines to produce intracranial xenograft and syngraft brain tumors in NSG or C57BL/6 mice, respectively. High-parameter flow cytometry and tissue immunofluorescence staining were used to characterize the immune cell compartment in each model. We investigated brain, spleen, lymph node, and bone marrow for the frequencies of different immune cells. Further, we investigated the effect of sub-lethal whole-body irradiation, commonly used for lymphodepletion, on immune cells in immunocompetent mice. ResultsIn our study of non-tumor-bearing mice, we observed dramatic differences between NSG and C57BL/6 mice, not only of T and B cells but also other immune cells, including decreased monocytes, dendritic cells, and eosinophils. In immunocompetent brain tumor-bearing mice, we observed significant differences in the numbers of immune cells compared to non-tumour-bearing controls, primarily in the brain; however, significant differences were also in the spleen, lymph node and bone marrow, particularly in the subsets of monocytes, T cells and dendritic cells. Sub-lethal irradiation caused lasting changes in immune composition. ConclusionsOur quantitative characterization of different immune compartments in orthotopic murine models of glioblastoma may allow for a better-informed selection of tumor models that are appropriate for the pre-clinical investigation of immunotherapies for glioblastoma.

cancer biology↗

bioRxiv2023

Endogenous bystander killing mechanisms enhance the activity of novel FAP-CAR-T cells against glioblastoma

ObjectivesCAR-T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR-T cells targeting FAP have never been investigated as a therapy for glioblastoma. MethodsWe generated a novel FAP targeting CAR with CD3{zeta} and CD28 signaling domains and tested the resulting CAR-T cells for their lytic activity and cytokine secretion function in vitro (using real-time impedance, flow cytometry, imaging, and bead-based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma). ResultsFAP-CAR-T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient-derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co-culture assays, we confirmed FAP-CAR-T cells mediate bystander killing of antigen-negative tumor cells, but only after activation by FAP-positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL-2 which activated the non-transduced fraction of the CAR-T product. Finally, a low dose of intravenously administered FAP-CAR-T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive glioblastoma cells. ConclusionsOur findings advance FAP as a leading candidate for clinical CAR-T therapy of glioblastoma and highlight under-recognized antigen non-specific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.

immunology↗

bioRxiv2022

Preclinical assessment of Fibroblast Activation Protein-specific MRI as an alternative to PSMA for image-guided focal therapy of prostate cancer

Accurate and precise delineation of gross tumour volumes remains a barrier to radiotherapy dose escalation and boost dosing in the treatment of solid tumours, such as prostate cancer. Magnetic resonance imaging of tumour molecular targets has the power to enable focal dose boosting, particularly when combined with technological advances such as MRI-LINAC. Fibroblast activation protein (FAP) is a transmembrane protein overexpressed in stromal components of >90% of epithelial carcinomas. Herein we compare targeted MRI of gold standard PSMA with FAP in the delineation of orthotopic tumours in a mouse model of prostate cancer. Control (no ligand), FAP and PSMA-targeting iron oxide nanoparticles were prepared with modification of an MRI agent (FerroTrace). Mice with orthotopic LNCaP tumours underwent T2-weighted 3D MRI 24 hours after intravenous injection of contrast agents. FAP and PSMA nanoparticles produced contrast enhancement on MRI when compared to control nanoparticles, which was most pronounced on the tumour periphery. FAP-targeted MRI increased the proportion of tumour contrast enhancing black pixels by 13.37% when compared to PSMA. Furthermore, analysis of changes in R2 values between healthy prostates and LNCaP tumours indicated an increase in contrast enhancing pixels in the tumour border of 15%, when targeting FAP, in contrast to PSMA This study demonstrates preclinical feasibility of PSMA and FAP-targeted MRI which can enable targeted image-guided focal therapy of localized prostate cancer.

bioengineering↗

bioRxiv2022

GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma

BackgroundAggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of CAR-T cell therapy have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T cell therapy also remain limited. MethodsWe profiled an extensive biobank of patients biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR-T cells from peripheral blood of glioblastoma and diffuse midline glioma patients and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. ResultsHere we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite glioblastoma patients having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an IL-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Markers associated with tumor control included those related to T-cell homing, infiltration, and cytotoxicity. ConclusionsTargeting GD2 using a clinically deployed CAR-T therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors. What is already known on this topicGD2 is a tumor antigen of significant interest for targeting immunotherapy. A single preclinical study has shown the effectiveness of GD2-CAR-T cell therapy in an orthotopic xenograft model of diffuse midline glioma. Similarly, there is one previous preclinical study of GD2-CAR-T therapy in a orthotopic glioblastoma xenograft model but tumor control was achieved only following intracranial injection of CAR-T cells. Given that GD2-CAR-T therapy is already being evaluated clinically for other tumor indications, it is important to establish whether there is an acceptable rationale for its use in brain tumors. What this study addsThis is the first description of a GD2-targeted CAR-T cell therapy that shows antitumor effectiveness in a preclinical model of human glioblastoma following intravenous administration. It is also the first study to investigate the potential effects that the immune profile of glioblastoma patients may have on the feasibility of CAR-T cell manufacturing. How this study might affect research, practice, or policyThe results of this study have led to the initiation of an Australian phase 1 clinical trial program aiming to test GD2-specific CAR-T cells for the treatment of childhood and adult primary brain tumors. The study provides valuable insights into the microenvironmental factors that influence the effectiveness of CAR-T cell therapy for this type of tumor, paving the way for further optimization of CAR-T cell technology for treatment of aggressive primary brain tumors such as glioblastoma.

immunology↗