Glioblastoma extracellular vesicles modulate immune PD-L1 expression in accessory macrophages upon radiotherapy.

Glioblastoma (GBM) is the most aggressive brain tumor, presenting major challenges due to limited treatment options. Standard care includes radiation therapy (RT) to curb tumor growth and alleviate symptoms, but its impact on GBM is limited. In this study, we investigated the effect of RT on immune suppression and whether extracellular vesicles (EVs) originating from GBM and taken up by the tumor microenvironment (TME) contribute to the induced therapeutic resistance. We observed that (1) ionizing radiation increases immune-suppressive markers on GBM cells, (2) macrophages exacerbate immune suppression in the TME by increasing PD-L1 in response to EVs derived from GBM cells which is further modulated by RT, and (3) RT increases CD206-positive macrophages which have the most potential in inducing a pro-oncogenic environment due to their increased uptake of tumor-derived EVs. In conclusion, RT affects GBM resistance by immuno-modulating EVs taken up by myeloid cells in the TME.

Arming AAV9 with a Single-Chain Fragment Variable Antibody Against PD-1 for Systemic Glioblastoma Therapy.

Glioblastoma (GBM) is a highly aggressive brain cancer with a low survival rate, prompting the exploration of novel therapeutic strategies. Immune checkpoint inhibitors have shown promise in cancer treatment but are associated with immune-related toxicities and brain penetration. Here, we present a targeted approach using an adeno-associated virus serotype 9 (AAV9) to systemically deliver a single-chain fragment variable antibody against PD-1 (scFv-PD-1) into the tumor microenvironment (TME). Single-cell RNA sequencing analysis revealed robust PD-1 expression in GBM TME, predominantly on T cells. AAV9-scFv-PD-1 expressed and secreted scFv-PD-1, which effectively binds to PD-1. Systemic administration of AAV9-scFv-PD-1 in an immunocompetent GBM mouse model resulted in a robust cytolytic T-cell activation at the tumor site, marked by accumulation of IFN-γ and Granzyme B, leading to a significant reduction in tumor growth. Importantly, AAV9-scFv-PD-1 treatment conferred a survival benefit, highlighting its therapeutic potential. This study demonstrates the feasibility of systemically delivered AAV9-mediated local expression of scFv-PD-1 for targeted immunotherapy in GBM and warrants further investigation for clinical translation.

Detection and localization of early- and late-stage cancers using platelet RNA.

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I-IV cancer patients and in half of 352 stage I-III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening.

Olfactory receptor 5B21 drives breast cancer metastasis.

Olfactory receptors (ORs), responsible for the sense of smell, play an essential role in various physiological processes outside the nasal epithelium, including cancer. In breast cancer, however, the expression and function of ORs remain understudied. We examined the significance of OR transcript abundance in primary and metastatic breast cancer to the brain, bone, and lung. Although 20 OR transcripts were differentially expressed in distant metastases, OR5B21 displayed an increased transcript abundance in all three metastatic sites compared with the primary tumor. Knockdown of OR5B21 significantly decreased the invasion and migration of breast cancer cells as well as metastasis to different organs especially the brain, whereas increasing of OR5B21 transcript abundance had the opposite effect. Mechanistically, OR5B21 expression was associated with epithelial to mesenchymal transition through the STAT3/NF-κB/CEBPß signaling axis. We propose OR5B21 (and potentially other ORs) as a novel oncogene contributing to breast cancer metastasis and a potential target for adjuvant therapy.

Small but Fierce: Tracking the Role of Extracellular Vesicles in Glioblastoma Progression and Therapeutic Resistance.

Glioblastoma is the most common and aggressive brain tumor in adults. Most patients die within a year and long-term survival remains rare, owing to a combination of rapid progression/degeneration, lack of successful treatments, and high recurrence rates. Extracellular vesicles are cell-derived membranous structures involved in numerous physiological and pathological processes. In the context of cancer, these biological nanoparticles play an important role in intercellular communication, allowing cancer cells to exchange information with each other, the tumor microenvironment as well as distant cells. Here, light is shed on the role of extracellular vesicles in glioblastoma heterogeneity, tumor microenvironment interactions, and therapeutic resistance, and an overview on means to track their release, uptake, and cargo delivery is provided.

Extracellular Vesicles Induce Mesenchymal Transition and Therapeutic Resistance in Glioblastomas through NF-κB/STAT3 Signaling.

Glioblastoma (GBM) is the most common primary malignant brain tumor and despite optimal treatment, long-term survival remains uncommon. GBM can be roughly divided into three different molecular subtypes, each varying in aggressiveness and treatment resistance. Recent evidence shows plasticity between these subtypes in which the proneural (PN) glioma stem-like cells (GSCs) undergo transition into the more aggressive mesenchymal (MES) subtype, leading to therapeutic resistance. Extracellular vesicles (EVs) are membranous structures secreted by nearly every cell and are shown to play a key role in GBM progression by acting as multifunctional signaling complexes. Here, it is shown that EVs derived from MES cells educate PN cells to increase stemness, invasiveness, cell proliferation, migration potential, aggressiveness, and therapeutic resistance by inducing mesenchymal transition through nuclear factor-κB/signal transducer and activator of transcription 3 signaling. The findings could potentially help explore new treatment strategies for GBM and indicate that EVs may also play a role in mesenchymal transition of different tumor types.

Blood platelet RNA enables the detection of multiple sclerosis.

BACKGROUND: In multiple sclerosis (MS), clinical assessment, MRI and cerebrospinal fluid are important in the diagnostic process. However, no blood biomarker has been confirmed as a useful tool in the diagnostic work-up. OBJECTIVES: Blood platelets contain a rich spliced mRNA repertoire that can alter during megakaryocyte development but also during platelet formation and platelet circulation. In this proof of concept study, we evaluate the diagnostic potential of spliced blood platelet RNA for the detection of MS. METHODS: We isolated and sequenced platelet RNA of blood samples obtained from 57 MS patients and 66 age- and gender-matched healthy controls (HCs). 60% was used to develop a particle swarm-optimized (PSO) support vector machine classification algorithm. The remaining 40% served as an independent validation series. RESULTS: In total, 1249 RNAs with differential spliced junction expression levels were identified between platelets of MS patients as compared to HCs, including EPSTI1, IFI6, and RPS6KA3, in line with reported inflammatory signatures in the blood of MS patients. The RNAs were subsequently used as input for a MS classifier, capable of detecting MS with 80% accuracy in the independent validation series. CONCLUSIONS: Spliced platelet RNA may enable the blood-based diagnosis of MS, warranting large-scale validation.

Patient-Derived Glioma Models: From Patients to Dish to Animals.

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults associated with a poor survival. Current standard of care consists of surgical resection followed by radiation and chemotherapy. GBMs are highly heterogeneous, having a complex interaction among different cells within the tumor as well as the tumor microenvironment. One of the main challenges in the neuro-oncology field in general, and GBM in particular, is to find an optimum culture condition that maintains the molecular genotype and phenotype as well as heterogeneity of the original tumor in vitro and in vivo. Established cell lines were shown to be a poor model of the disease, failing to recapitulate the phenotype and harboring non-parental genotypic mutations. Given the growing understanding of GBM biology, the discovery of glioma cancer stem-like cells (GSCs), and their role in tumor formation and therapeutic resistance, scientists are turning more towards patient-derived cells and xenografts as a more representative model. In this review, we will discuss the current state of patient-derived GSCs and their xenografts; and provide an overview of different established models to study GBM biology and to identify novel therapeutics in the pre-clinical phase.

Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles.

Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.

TRPM8 Activation via 3-Iodothyronamine Blunts VEGF-Induced Transactivation of TRPV1 in Human Uveal Melanoma Cells.

In human uveal melanoma (UM), tumor enlargement is associated with increases in aqueous humor vascular endothelial growth factor-A (VEGF-A) content that induce neovascularization. 3-Iodothyronamine (3-T1AM), an endogenous thyroid hormone metabolite, activates TRP melastatin 8 (TRPM8), which blunts TRP vanilloid 1 (TRPV1) activation by capsaicin (CAP) in human corneal, conjunctival epithelial cells, and stromal cells. We compare here the effects of TRPM8 activation on VEGF-induced transactivation of TRPV1 in an UM cell line (92.1) with those in normal primary porcine melanocytes (PM) since TRPM8 is upregulated in melanoma. Fluorescence Ca2+-imaging and planar patch-clamping characterized functional channel activities. CAP (20 µM) induced Ca2+ transients and increased whole-cell currents in both the UM cell line and PM whereas TRPM8 agonists, 100 µM menthol and 20 µM icilin, blunted such responses in the UM cells. VEGF (10 ng/ml) elicited Ca2+ transients and augmented whole-cell currents, which were blocked by capsazepine (CPZ; 20 µM) but not by a highly selective TRPM8 blocker, AMTB (20 µM). The VEGF-induced current increases were not augmented by CAP. Both 3-T1AM (1 µM) and menthol (100 µM) increased the whole-cell currents, whereas 20 µM AMTB blocked them. 3-T1AM exposure suppressed both VEGF-induced Ca2+ transients and increases in underlying whole-cell currents. Taken together, functional TRPM8 upregulation in UM 92.1 cells suggests that TRPM8 is a potential drug target for suppressing VEGF induced increases in neovascularization and UM tumor growth since TRPM8 activation blocked VEGF transactivation of TRPV1.