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Abstract: Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.
Keywords: malignant gliomas, cancer stem cells, nanobodies
Published in DiRROS: 02.08.2024; Views: 90; Downloads: 69
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Abstract: Background: High-grade serous carcinoma (HGSC) is often associated with ascites at presentation. Our objective was to quantify immune cells (ICs) in ascites prior to any treatment was given and evaluate their impact on progression-free survival (PFS) and overall survival (OS). Patients and methods: Forty-seven patients with primary HGSC and ascites were included. Flow-cytometric analysis was performed to detect percentages of CD3+ T cells (CD4+, CD8+, Tregs, and NKT cells), B cells, NK cells (CD56brightCD16- and CD56dimCD16+ subsets), macrophages and dendritic cells (DCs). Furthermore, CD103 expression was analyzed on T cells and their subsets, while PD-1 and PD-L1 expression on all ICs. Cut-off of low and high percentages of ICs was determined by the median of variables, and correlation with PFS and OS was calculated. Results: CD3+ cells were the predominant ICs (median 51%), while the presence of other ICs was much lower (median ≤10%). CD103+ expression was mostly present on CD8+, and not CD4+ cells. PD-1 was mainly expressed on CD3+ T cells (median 20%), lower expression was observed on other ICs (median ≤10%). PD-L1 expression was not detected. High percentages of CD103+CD3+ T cells, PD-1+ Tregs, CD56brightCD16- NK cells, and DCs correlated with prolonged PFS and OS, while high percentages of CD8+ cells, macrophages, and PD-1+CD56brightCD16- NK cells, along with low percentages of CD4+ cells, correlated with better OS only. DCs were the only independent prognostic marker among all ICs. Conclusions: Our results highlight the potential of ascites tumor-immune microenvironment to provide additional prognostic information for HGSC patients. However, a larger patient cohort and longer follow-up are needed to confirm our finBackground: High-grade serous carcinoma (HGSC) is often associated with ascites at presentation. Our objective was to quantify immune cells (ICs) in ascites prior to any treatment was given and evaluate their impact on progression-free survival (PFS) and overall survival (OS). Patients and methods: Forty-seven patients with primary HGSC and ascites were included. Flow-cytometric analysis was performed to detect percentages of CD3+ T cells (CD4+, CD8+, Tregs, and NKT cells), B cells, NK cells (CD56brightCD16- and CD56dimCD16+ subsets), macrophages and dendritic cells (DCs). Furthermore, CD103 expression was analyzed on T cells and their subsets, while PD-1 and PD-L1 expression on all ICs. Cut-off of low and high percentages of ICs was determined by the median of variables, and correlation with PFS and OS was calculated. Results: CD3+ cells were the predominant ICs (median 51%), while the presence of other ICs was much lower (median ≤10%). CD103+ expression was mostly present on CD8+, and not CD4+ cells. PD-1 was mainly expressed on CD3+ T cells (median 20%), lower expression was observed on other ICs (median ≤10%). PD-L1 expression was not detected. High percentages of CD103+CD3+ T cells, PD-1+ Tregs, CD56brightCD16- NK cells, and DCs correlated with prolonged PFS and OS, while high percentages of CD8+ cells, macrophages, and PD-1+CD56brightCD16- NK cells, along with low percentages of CD4+ cells, correlated with better OS only. DCs were the only independent prognostic marker among all ICs. Conclusions: Our results highlight the potential of ascites tumor-immune microenvironment to provide additional prognostic information for HGSC patients. However, a larger patient cohort and longer follow-up are needed to confirm our findings.dings.
Keywords: high-grade serous carcinoma, immune cells, prognostic markers
Published in DiRROS: 26.07.2024; Views: 260; Downloads: 107
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Abstract: The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment.
Keywords: biomarker, CD9, glioblastoma stem cells, neural stem cells, tetraspanin
Published in DiRROS: 26.07.2024; Views: 110; Downloads: 83
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Keywords: chimeric antigen receptor, adoptive cell therapy, cancer, cellular immunotherapy, gene-engineered immune cells
Published in DiRROS: 25.07.2024; Views: 93; Downloads: 90
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Abstract: Glioblastoma (GBM) is the most aggressive primary brain tumor with poor patient survival that is at least partly caused by malignant and therapy-resistant glioma stem-like cells (GSLCs) that are protected in GSLC niches. Previously, we have shown that the chemo-attractant stromal-derived factor-1α (SDF-1α), its C-X-C receptor type 4 (CXCR4) and the cysteine protease cathepsin K (CatK) are localized in GSLC niches in glioblastoma. Here, we investigated whether SDF-1α is a niche factor that through its interactions with CXCR4 and/or its second receptor CXCR7 on GSLCs facilitates their homing to niches. Furthermore, we aimed to prove that SDF-1α cleavage by CatK inactivates SDF-1α and inhibits the invasion of GSLCs. We performed mass spectrometric analysis of cleavage products of SDF-1α after proteolysis by CatK. We demonstrated that CatK cleaves SDF-1α at 3 sites in the N-terminus, which is the region of SDF-1α that binds to its receptors. Confocal imaging of human GBM tissue sections confirmed co-localization of SDF-1α and CatK in GSLC niches. In accordance, 2D and 3D invasion experiments using CXCR4/CXCR7-expressing GSLCs and GBM cells showed that SDF-1α had chemotactic activity whereas CatK cleavage products of SDF-1α did not. Besides, CXCR4 inhibitor plerixafor inhibited invasion of CXCR4/CXCR7-expressing GSLCs. In conclusion, CatK can cleave and inactivate SDF-1α. This implies that CatK activity facilitates migration of GSLCs out of niches. We propose that activation of CatK may be a promising strategy to prevent homing of GSLCs in niches and thus render these cells sensitive to chemotherapy and radiation.
Keywords: glioma stem-like cells, niche, stromal derived factor-[alpha], cathepsin K
Published in DiRROS: 24.07.2024; Views: 101; Downloads: 112
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