Co-expression of TIMP-1 and its cell surface binding partner CD63 in glioblastomas.

BACKGROUND: We have previously identified tissue inhibitor of metalloproteinases-1 (TIMP-1) as a prognostic marker in glioblastomas. TIMP-1 has been associated with chemotherapy resistance, and CD63, a known TIMP-1-binding protein, has been suggested to be responsible for this effect. The aim of this study was to assess CD63 expression in astrocytomas focusing on the prognostic potential of CD63 alone and in combination with TIMP-1. METHODS: CD63 expression was investigated immunohistochemically in a cohort of 111 astrocytomas and correlated to tumor grade and overall survival by semi-quantitative scoring. CD63 expression in tumor-associated microglia/macrophages was examined by double-immunofluorescence with ionized calcium-binding adapter molecule 1 (Iba1). The association between CD63 and TIMP-1 was investigated using previously obtained TIMP-1 data from our astrocytoma cohort. Cellular co-expression of TIMP-1 and CD63 as well as TIMP-1 and the tumor stem cell-related markers CD133 and Sox2 was investigated with immunofluorescence. TIMP-1 and CD63 protein interaction was detected by an oligonucleotide-based proximity ligation assay and verified using co-immunoprecipitation. RESULTS: The expression of CD63 was widely distributed in astrocytomas with a significantly increased level in glioblastomas. CD63 levels did not significantly correlate with patient survival at a protein level, and CD63 did not augment the prognostic significance of TIMP-1. Up to 38% of the CD63+ cells expressed Iba1; however, Iba1 did not appear to impact the prognostic value of CD63. A significant correlation was found between TIMP-1 and CD63, and the TIMP-1 and CD63 proteins were co-expressed at the cellular level and located in close molecular proximity, suggesting that TIMP-1 and CD63 could be co-players in glioblastomas. Some TIMP-1+ cells expressed CD133 and Sox2. CONCLUSION: The present study suggests that CD63 is highly expressed in glioblastomas and that TIMP-1 and CD63 interact. CD63 does not add to the prognostic value of TIMP-1. Co-expression of TIMP-1 and stem cell markers as well as the wide expression of CD63 might suggest a role for TIMP-1 and CD63 in glioblastoma stemness.

Surgical resection of glioblastomas induces pleiotrophin-mediated self-renewal of glioblastoma stem cells in recurrent tumors.

BACKGROUND: Glioblastomas are highly resistant to therapy, and virtually all patients experience tumor recurrence after standard-of-care treatment. Surgical tumor resection is a cornerstone in glioblastoma therapy, but its impact on cellular phenotypes in the local postsurgical microenvironment has yet to be fully elucidated. METHODS: We developed a preclinical orthotopic xenograft tumor resection model in rats with integrated 18F-FET PET/CT imaging. Primary and recurrent tumors were subject to bulk and single-cell RNA sequencing. Differentially expressed genes and pathways were investigated and validated using tissue specimens from the xenograft model, 23 patients with matched primary/recurrent tumors, and a cohort including 190 glioblastoma patients. Functional investigations were performed in vitro with multiple patient-derived cell cultures. RESULTS: Tumor resection induced microglia/macrophage infiltration, angiogenesis as well as proliferation and upregulation of several stem cell-related genes in recurrent tumor cells. Expression changes of selected genes SOX2, POU3F2, OLIG2, and NOTCH1 were validated at the protein level in xenografts and early recurrent patient tumors. Single-cell transcriptomics revealed the presence of distinct phenotypic cell clusters in recurrent tumors which deviated from clusters found in primary tumors. Recurrent tumors expressed elevated levels of pleiotrophin (PTN), secreted by both tumor cells and tumor-associated microglia/macrophages. Mechanistically, PTN could induce tumor cell proliferation, self-renewal, and the stem cell program. In glioblastoma patients, high PTN expression was associated with poor overall survival and identified as an independent prognostic factor. CONCLUSION: Surgical tumor resection is an iatrogenic driver of PTN-mediated self-renewal in glioblastoma tumor cells that promotes therapeutic resistance and tumor recurrence.

Expression and Prognostic Value of the Immune Checkpoints Galectin-9 and PD-L1 in Glioblastomas.

Immunotherapeutic targeting of the PD-1/PD-L1 axis has been widely implemented for treatment of several cancer types but shown disappointing results in glioblastomas (GBMs), potentially due to compensatory mechanisms of other expressed immune checkpoints. Galectin-9 is an immune-checkpoint protein that facilitates T-cell exhaustion and apoptosis and could be a potential target for immune-checkpoint inhibition. A total of 163 GBMs IDH wildtype were immunostained with anti-Galectin-9 and PD-L1 antibodies. Software-based quantitation of immunostainings was performed and co-expression was investigated using double immunofluorescence. Both Galectin-9 and PD-L1 protein expression were found in all 163 tumors and showed a significant positive correlation (p = 0.0017). Galectin-9 expression varied from 0.01% to 32% (mean = 6.61%), while PD-L1 membrane expression ranged from 0.003% to 0.14% (mean = 0.048%) of total tumor area. Expression of Galectin-9 and PD-L1 was found on both microglia/macrophages and tumor cells, and colocalization of both markers was found in 88.3% of tumors. In multivariate analysis, neither Galectin-9 (HR = 0.99), PD-L1 (HR = 1.05), nor their combinations showed prognostic value. Galectin-9 and PD-L1 were expressed in all investigated GBMs and the majority of patients had co-expression, which may provide rationale for multi-targeted immune checkpoint inhibition.

The multi-target small-molecule inhibitor SB747651A shows in vitro and in vivo anticancer efficacy in glioblastomas.

Glioblastoma multiforme is the most common primary brain tumor and among the most lethal types of cancer. Several mono-target small molecule-inhibitors have been investigated as novel therapeutics, thus far with poor success. In this study we investigated the anticancer effects of SB747651A, a multi-target small-molecule inhibitor, in three well characterized patient-derived glioblastoma spheroid cultures and a murine orthotopic xenograft model. Concentrations of 5-10 µM SB747651A reduced cell proliferation, spheroid formation, migration and chemoresistance, while apoptotic cell death increased. Investigation of oncogenic kinase signaling showed decreased phosphorylation levels of mTOR, CREB, GSK3 and GYS1 leading to altered glycogen metabolism and formation of intracellular reactive oxygen species. Expression levels of cancer stemness marker SOX2 were reduced in treated tumor cells and SB747651A treatment significantly prolonged survival of mice with intracranial glioblastoma xenografts, while no adverse effects were observed in vivo at doses of 25 mg/kg administered 5 days/week for 8 weeks. These findings suggest that SB747651A has anticancer effects in glioblastoma. The cancer-related pathophysiological mechanisms targeted by SB747651A are shared among many types of cancer; however, an in-depth clarification of the mechanisms of action in cancer cells is important before further potential application of SB747651A as an anticancer agent can be considered.

Asymmetric cell division promotes therapeutic resistance in glioblastoma stem cells.

Asymmetric cell division (ACD) enables the maintenance of a stem cell population while simultaneously generating differentiated progeny. Cancer stem cells (CSCs) undergo multiple modes of cell division during tumor expansion and in response to therapy, yet the functional consequences of these division modes remain to be determined. Using a fluorescent reporter for cell surface receptor distribution during mitosis, we found that ACD generated a daughter cell with enhanced therapeutic resistance and increased coenrichment of EGFR and neurotrophin receptor (p75NTR) from a glioblastoma CSC. Stimulation of both receptors antagonized differentiation induction and promoted self-renewal capacity. p75NTR knockdown enhanced the therapeutic efficacy of EGFR inhibition, indicating that coinheritance of p75NTR and EGFR promotes resistance to EGFR inhibition through a redundant mechanism. These data demonstrate that ACD produces progeny with coenriched growth factor receptors, which contributes to the generation of a more therapeutically resistant CSC population.

Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches.

Glioblastoma (GBM) displays marked cellular and metabolic heterogeneity that varies among cellular microenvironments within a tumor. Metabolic targeting has long been advocated as a therapy against many tumors including GBM, but how lipid metabolism is altered to suit different microenvironmental conditions and whether cancer stem cells (CSCs) have altered lipid metabolism are outstanding questions in the field. We interrogated gene expression in separate microenvironments of GBM organoid models that mimic the transition between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones using spatial-capture RNA-sequencing. We revealed a striking difference in lipid processing gene expression and total lipid content between diverse cell populations from the same patient, with lipid enrichment in hypoxic organoid cores and also in perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and pseudopalisading regions of clinical GBM specimens, but not lower-grade brain tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis of multiple patient-derived models revealed a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs, but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression which was essential to maintain CSC viability and self-renewal. Our data demonstrate spatially and hierarchically distinct lipid metabolism phenotypes occur clinically in the majority of patients, can be recapitulated in laboratory models, and may represent therapeutic targets for GBM.

Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas.

Most glioblastoma patients have a dismal prognosis, although some survive several years. However, only few biomarkers are available to predict the disease course. EGR1 and EGR3 have been linked to glioblastoma stemness and tumour progression, and this study aimed to investigate their spatial expression and prognostic value in gliomas. Overall 207 gliomas including 190 glioblastomas were EGR1/EGR3 immunostained and quantified. A cohort of 21 glioblastomas with high P53 expression and available tissue from core and periphery was stained with double-immunofluorescence (P53-EGR1 and P53-EGR3) and quantified.EGR1 expression increased with WHO-grade, and declined by 18.9% in the tumour periphery vs. core (P = 0.01), while EGR3 expression increased by 13.8% in the periphery vs. core (P = 0.04). In patients with high EGR1 expression, 83% had methylated MGMT-promoters, while all patients with low EGR1 expression had un-methylated MGMT-promoters. High EGR3 expression in MGMT-methylated patients was associated with poor survival (HR = 1.98; 95%CI 1.22-3.22; P = 0.006), while EGR1 high/EGR3 high, was associated with poor survival vs. EGR1 high/EGR3 low (HR = 2.11; 95%CI 1.25-3.56; P = 0.005). EGR1 did not show prognostic value, but could be involved in MGMT-methylation. Importantly, EGR3 may be implicated in cell migration, while its expression levels seem to be prognostic in MGMT-methylated patients.

AN1-type zinc finger protein 3 (ZFAND3) is a transcriptional regulator that drives Glioblastoma invasion.

The infiltrative nature of Glioblastoma (GBM), the most aggressive primary brain tumor, critically prevents complete surgical resection and masks tumor cells behind the blood brain barrier reducing the efficacy of systemic treatment. Here, we use a genome-wide interference screen to determine invasion-essential genes and identify the AN1/A20 zinc finger domain containing protein 3 (ZFAND3) as a crucial driver of GBM invasion. Using patient-derived cellular models, we show that loss of ZFAND3 hampers the invasive capacity of GBM, whereas ZFAND3 overexpression increases motility in cells that were initially not invasive. At the mechanistic level, we find that ZFAND3 activity requires nuclear localization and integral zinc-finger domains. Our findings indicate that ZFAND3 acts within a nuclear protein complex to activate gene transcription and regulates the promoter of invasion-related genes such as COL6A2, FN1, and NRCAM. Further investigation in ZFAND3 function in GBM and other invasive cancers is warranted.

Short-term treatment with oral amiodarone resulting in bilateral optic neuropathy and permanent blindness.

A 66-year-old man was hospitalised due to worsening of his atrial fibrillation, and successfully underwent an electrocardioversion to restore sinus rhythm. He was discharged with a prescription of 600 mg amiodarone daily to retain the sinus rhythm prospectively. 3 weeks after discharge, he noticed that his vision became blurry with cloudy areas spreading throughout his field of view on both eyes simultaneously. The visual symptoms gradually worsened throughout the following 5 months, and the patient was hospitalised in the department of ophthalmology. At this point, his vision had dramatically decreased and visual acuity was measured to 0.05 (1.3 LogMAR) on both eyes. After a thorough medical examination, the patient was diagnosed with an amiodarone-induced toxic optic neuropathy. Regular follow-ups conducted throughout 7 years have shown that the patient's vision has stabilised, but remains limited to the ability of recognising hand motions on both eyes, thus characterising him as blind.