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Infiltrating CTLs in human glioblastoma establish immunological synapses with tumorigenic cells.

ABSTRACT: The immunological synapse between T cells and tumor cells is believed to be important for effective tumor clearance. However, the immunological synapse has never been imaged or analyzed in detail in human tissue. In this work, intercellular interactions between T cells and tumor cells were analyzed in detail in human glioblastoma. After characterization of the population of infiltrating T cells by multiple immunofluorescence staining and stereological quantification, the microanatomy of T cell-tumor cell intercellular communication was analyzed in detail using confocal microscopy and three-dimensional rendering. Cytotoxic T lymphocytes that infiltrated human glioblastoma underwent rearrangement when in contact with tumor cells, to form a three-dimensional structure in the intercellular contact area; this was characterized by microclusters of the CD3/TCR complex, re-arrangement of the cytoskeleton, and granzyme B polarization. In addition, such T cell-targeted cells show fragmentation of the microtubular system and increased expression levels of cleaved caspase 3, which suggests that cytotoxic T lymphocytes likely provoke changes in tumor cells and subsequently induce cell death. These results show that the formation of the cytotoxic T lymphocyte immunological synapse occurs in human tissue and may be relevant for the effective immune-mediated clearance of tumorigenic cells, therefore opening up new avenues for glioblastoma immunotherapy.

SUBMITTER: Barcia C

PROVIDER: S-EPMC2716973 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Infiltrating CTLs in human glioblastoma establish immunological synapses with tumorigenic cells.

Barcia Carlos C Gómez Aurora A Gallego-Sanchez José M JM Perez-Vallés Ana A Castro Maria G MG Lowenstein Pedro R PR Barcia Carlos C Herrero Maria-Trinidad MT

The American journal of pathology 20090723 2

The immunological synapse between T cells and tumor cells is believed to be important for effective tumor clearance. However, the immunological synapse has never been imaged or analyzed in detail in human tissue. In this work, intercellular interactions between T cells and tumor cells were analyzed in detail in human glioblastoma. After characterization of the population of infiltrating T cells by multiple immunofluorescence staining and stereological quantification, the microanatomy of T cell-t ...[more]

PMID: 19628762

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Project description:The identification and significance of cancer stem-like cells in malignant gliomas remains controversial. It has been proposed that cancer stem-like cells display increased drug resistance, through the expression of ATP-binding cassette transporters that detoxify cells by effluxing exogenous compounds. Here, we investigated the 'side population' phenotype based on efflux properties of ATP-binding cassette transporters in freshly isolated human glioblastoma samples and intracranial xenografts derived thereof. Using fluorescence in situ hybridization analysis on sorted cells obtained from glioblastoma biopsies, as well as human tumour xenografts developed in immunodeficient enhanced green fluorescence protein-expressing mice that allow an unequivocal tumour-stroma discrimination, we show that side population cells in human glioblastoma are non-neoplastic and exclusively stroma-derived. Tumour cells were consistently devoid of efflux properties regardless of their genetic background, tumour ploidy or stem cell associated marker expression. Using multi-parameter flow cytometry we identified the stromal side population in human glioblastoma to be brain-derived endothelial cells with a minor contribution of astrocytes. In contrast with their foetal counterpart, neural stem/progenitor cells in the adult brain did not display the side population phenotype. Of note, we show that CD133-positive cells often associated with cancer stem-like cells in glioblastoma biopsies, do not represent a homogenous cell population and include CD31-positive endothelial cells. Interestingly, treatment of brain tumours with the anti-angiogenic agent bevacizumab reduced total vessel density, but did not affect the efflux properties of endothelial cells. In conclusion our findings contribute to an unbiased identification of cancer stem-like cells and stromal cells in brain neoplasms, and provide novel insight into the complex issue of drug delivery to the brain. Since efflux properties of endothelial cells are likely to compromise drug availability, transiently targeting ATP-binding cassette transporters may be a valuable therapeutic strategy to improve treatment effects in brain tumours.

Project description:BackgroundGlioblastoma (GBM) is the most common primary brain malignancy and confers a dismal prognosis. GBMs harbor glioblastoma-initiating cells (GICs) that drive tumorigenesis and contribute to therapeutic resistance and tumor recurrence. Consequently, there is a strong rationale to target this cell population in order to develop new molecular therapies against GBM. Accumulating evidence indicates that Nα-terminal acetyltransferases (NATs), that are dysregulated in numerous human cancers, can serve as therapeutic targets.MethodsMicroarrays were used to study the expression of several NATs including NAT12/NAA30 in clinical samples and stem cell cultures. The expression of NAT12/NAA30 was analyzed using qPCR, immunolabeling and western blot. We conducted shRNA-mediated knockdown of NAT12/NAA30 gene in GICs and studied the effects on cell viability, sphere-formation and hypoxia sensitivity. Intracranial transplantation to SCID mice enabled us to investigate the effects of NAT12/NAA30 depletion in vivo. Using microarrays we identified genes and biochemical pathways whose expression was altered upon NAT12/NAA30 down-regulation.ResultsWhile decreased expression of the distal 3'UTR of NAT12/NAA30 was generally observed in GICs and GBMs, this gene was strongly up-regulated at the protein level in GBM and GICs. The increased protein levels were not caused by increased levels of the steady state mRNA but rather by other mechanisms. Also, shorter 3'UTR of NAT12/NAA30 correlated with poor survival in glioma patients. As well, we observed previously not described nuclear localization of this typically cytoplasmic protein. When compared to non-silencing controls, cells featuring NAT12/NAA30 knockdown exhibited reduced cell viability, sphere-forming ability, and mitochondrial hypoxia tolerance. Intracranial transplantation showed that knockdown of NAT12/NAA30 resulted in prolonged animal survival. Microarray analysis of the knockdown cultures showed reduced levels of HIF1α and altered expression of several other genes involved in the hypoxia response. Furthermore, NAT12/NAA30 knockdown correlated with expressional dysregulation of genes involved in the p53 pathway, ribosomal assembly and cell proliferation. Western blot analysis revealed reduction of HIF1α, phospho-MTOR(Ser2448) and higher levels of p53 and GFAP in these cultures.ConclusionNAT12/NAA30 plays an important role in growth and survival of GICs possibly by regulating hypoxia response (HIF1α), levels of p-MTOR (Ser2448) and the p53 pathway.

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Infiltrating CTLs in human glioblastoma establish immunological synapses with tumorigenic cells.

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Infiltrating CTLs in human glioblastoma establish immunological synapses with tumorigenic cells.

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