NK cells with KIR2DS2 immunogenotype have a functional activation advantage to efficiently kill glioblastoma and prolong animal survival

Glioblastomas (GBMs) are lethal brain cancers that are resistant to current therapies. We investigated the cytotoxicity of human allogeneic NK cells against patient-derived GBM in vitro and in vivo, as well as mechanisms mediating their efficacy. We demonstrate that KIR2DS2 immunogenotype NK cells were more potent killers, notwithstanding the absence of inhibitory killer Ig–like receptor (KIR)-HLA ligand mismatch. FACS-sorted and enriched KIR2DS2+ NK cell subpopulations retained significantly high levels of CD69 and CD16 when in contact with GBM cells at a 1:1 ratio and highly expressed CD107a and secreted more soluble CD137 and granzyme A. In contrast, KIR2DS2− immunogenotype donor NK cells were less cytotoxic against GBM and K562, and, similar to FACS-sorted or gated KIR2DS2− NK cells, significantly diminished CD16, CD107a, granzyme A, and CD69 when in contact with GBM cells. Furthermore, NK cell–mediated GBM killing in vitro depended upon the expression of ligands for the activating receptor NKG2D and was partially abrogated by Ab blockade. Treatment of GBM xenografts in NOD/SCID mice with NK cells from a KIR2DS2+ donor lacking inhibitory KIR-HLA ligand mismatch significantly prolonged the median survival to 163 d compared with vehicle controls (log-rank test, p = 0.0001), in contrast to 117.5 d (log-rank test, p = 0.0005) for NK cells with several inhibitory KIR-HLA ligand mismatches but lacking KIR2DS2 genotype. Significantly more CD56+CD16+ NK cells from a KIR2DS2+ donor survived in nontumor-bearing brains 3 wk after infusion compared with KIR2DS2− NK cells, independent of their proliferative capacity. In conclusion, KIR2DS2 identifies potent alloreactive NK cells against GBM that are mediated by commensurate, but dominant, activating signals.publishedVersio

Tumour-associated glial host cells display a stem-like phenotype with a distinct gene expression profile and promote growth of GBM xenografts

Background: Little is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers. Methods: We isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b+ immune and CD31+ endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs. Results: TAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs. Conclusions: Our data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay.publishedVersio

Impact of hypoxia on the expression of cell line markers and behavior of cultured glioma cell lines

Glioblastoma multiforme (GBM) constitute over 50 % of primary brain tumors in adults. The tumor is heterogeneous and highly infiltrative. Its nature makes it extremely aggressive and incurable. Thus, today's treatment modalities only have a palliative function and provide a modest survival benefit. This states the need for further research to reveal the biological mechanisms behind GBM development. The name glioblastoma multiforme reflects the expression of markers of many different CNS cell lineages in this tumor. Notably, GBMs have been reported to comprise cells that express stem cell markers, and it is postulated that glioma stem cells is the cell type initiating and maintaining this tumor. Also, several lines of evidence strongly suggest that hypoxia is a cancer stem cell niche. Furthermore, hypoxia is associated with poor prognosis in many solid cancers, being implicated in induction of radio- and chemoresistance and in tumor invasion. Hence, hypoxia and cancer stem cells may be two factors that contribute to the failure to treat this tumor. They might therefore be candidates which can be targeted in new therapeutical strategies for GBM. To assess the expression of cancer stem cell markers in hypoxia, five glioma cell lines were cultured for various time periods in normoxic (21 %) and hypoxic (0.5 and 2 %) conditions. Expression of a panel of cell lineage markers was investigated in the different oxygen conditions. Additionally, cell behavior in hypoxia compared to normoxia was evaluated by investigating proliferation, migration and apoptosis. We found that none of the stem cell markers that we looked at were upregulated in hypoxia, which is contradictory to previous reports. Instead, these stem cell markers and the astrocytic marker GFAP were all downregulated. Furthermore, cell proliferation and migration both decreased in hypoxia. This points at an overall slow down of the glioma cell machinery in hypoxia. Other studies have mainly been performed on small selections of biopsy material cultured in serum free medium, and this approach should be included in the present study in the future

Fibroblasts in urothelial bladder cancer define stroma phenotypes that are associated with clinical outcome

Little attention was given to the interaction between tumor and stromal cells in urothelial bladder carcinoma (UBC). While recent studies point towards the existence of different fibroblast subsets, no comprehensive analyses linking different fibroblast markers to UBC patient survival have been performed so far. Through immunohistochemical analysis of five selected fibroblast markers, namely alpha smooth muscle actin (ASMA), CD90/Thy-1, fibroblast activation protein (FAP), platelet derived growth factor receptor-alpha and -beta (PDGFRa,-b), this study investigates their association with survival and histopathological characteristics in a cohort of 344 UBC patients, involving both, muscle-invasive and non-muscle-invasive cases. The data indicates that combinations of stromal markers are more suited to identify prognostic patient subgroups than single marker analysis. Refined stroma-marker-based patient stratification was achieved through cluster analysis and identified a FAP-dominant patient cluster as independent marker for shorter 5-year-survival (HR(95% CI)2.25(1.08-4.67), p = 0.030). Analyses of interactions between fibroblast and CD8a-status identified a potential minority of cases with CD90-defined stroma and high CD8a infiltration showing a good prognosis of more than 80% 5-year-survival. Presented analyses point towards the existence of different stroma-cell subgroups with distinct tumor-modulatory properties and motivate further studies aiming to better understand the molecular tumor-stroma crosstalk in UBC

NK cells with KIR2DS2 immunogenotype have a functional activation advantage to efficiently kill glioblastoma and prolong animal survival

Glioblastomas (GBMs) are lethal brain cancers that are resistant to current therapies. We investigated the cytotoxicity of human allogeneic NK cells against patient-derived GBM in vitro and in vivo, as well as mechanisms mediating their efficacy. We demonstrate that KIR2DS2 immunogenotype NK cells were more potent killers, notwithstanding the absence of inhibitory killer Ig–like receptor (KIR)-HLA ligand mismatch. FACS-sorted and enriched KIR2DS2+ NK cell subpopulations retained significantly high levels of CD69 and CD16 when in contact with GBM cells at a 1:1 ratio and highly expressed CD107a and secreted more soluble CD137 and granzyme A. In contrast, KIR2DS2− immunogenotype donor NK cells were less cytotoxic against GBM and K562, and, similar to FACS-sorted or gated KIR2DS2− NK cells, significantly diminished CD16, CD107a, granzyme A, and CD69 when in contact with GBM cells. Furthermore, NK cell–mediated GBM killing in vitro depended upon the expression of ligands for the activating receptor NKG2D and was partially abrogated by Ab blockade. Treatment of GBM xenografts in NOD/SCID mice with NK cells from a KIR2DS2+ donor lacking inhibitory KIR-HLA ligand mismatch significantly prolonged the median survival to 163 d compared with vehicle controls (log-rank test, p = 0.0001), in contrast to 117.5 d (log-rank test, p = 0.0005) for NK cells with several inhibitory KIR-HLA ligand mismatches but lacking KIR2DS2 genotype. Significantly more CD56+CD16+ NK cells from a KIR2DS2+ donor survived in nontumor-bearing brains 3 wk after infusion compared with KIR2DS2− NK cells, independent of their proliferative capacity. In conclusion, KIR2DS2 identifies potent alloreactive NK cells against GBM that are mediated by commensurate, but dominant, activating signals

Surgical debulking promotes recruitment of macrophages and triggers glioblastoma phagocytosis in combination with CD47 blocking immunotherapy

Surgical resection is a standard component of treatment in the clinical management of patients with glioblastoma multiforme (GBM). However, experimental therapies are rarely investigated in the context of tumor debulking in preclinical models. Here, a surgical debulking GBM xenograft model was developed in nude rats, and was used in combination with CD47 blocking immunotherapy, a novel treatment strategy that triggers phagocytosis of tumor cells by macrophages in diverse cancer types including GBM. Orthotopic patient–derived xenograft tumors expressing CD47 were resected at 4 weeks after implantation and immediately thereafter treated with anti-CD47 or control antibodies injected into the cavity. Debulking prolonged survival (median survival, 68.5 vs 42.5 days, debulking and non-debulking survival times, respectively; n = 6 animals/group; P = 0.0005). Survival was further improved in animals that underwent combination treatment with anti-CD47 mAbs (median survival, 81.5 days vs 69 days, debulking + anti-CD47 vs debulking + control IgG, respectively; P = 0.0007). Immunohistochemistical staining of tumor sections revealed an increase in recruitment of cells positive for CD68, a marker for macrophages/immune cell types, to the surgical site (50% vs 10%, debulking vs non-debulking, respectively). Finally, analysis of tumor protein lysates on antibody microarrays demonstrated an increase in pro-inflammatory cytokines, such as CXCL10, and a decrease in angiogenic proteins in debulking + anti-CD47 vs non-debulking + IgG tumors. The results indicated that surgical resection combined with anti-CD47 blocking immunotherapy promoted an inflammatory response and prolonged survival in animals, and is therefore an attractive strategy for clinical translation

Tumour-associated glial host cells display a stem-like phenotype with a distinct gene expression profile and promote growth of GBM xenografts

Background: Little is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers. Methods: We isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b+ immune and CD31+ endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs. Results: TAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs. Conclusions: Our data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay