Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification

Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-amplification of WTA products, quantification of amplified cDNA yields and final qPCR quantification, to identify the most reliable and accurate workflow for quantitation of gene expression of the ERBB2 gene in DCCs. We found that absolute quantification out-performs relative quantification. We then validated the performance of our method on single cells of established breast cancer cell lines displaying distinct levels of HER2 protein. The different protein levels were faithfully reflected by transcript expression across the tested cell lines thereby proving the accuracy of our approach. Finally, we applied our method to breast cancer DCCs of a patient undergoing anti-HER2-directed therapy. Here, we were able to measure ERBB2 expression levels in all HER2-protein-positive DCCs. In summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of ERBB2 in DCCs

TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma

Glioblastoma (GB) IDH-wildtype is the most malignant primary brain tumor. It is particularly resistant to current immunotherapies. Translocator protein 18 kDa (TSPO) is upregulated in GB and correlates with malignancy and poor prognosis, but also with increased immune infiltration. Here, we studied the role of TSPO in the regulation of immune resistance of human GB cells. The role of TSPO in tumor immune resistance was experimentally determined in primary brain tumor initiating cells (BTICs) and cell lines through genetic manipulation of TSPO expression and subsequent cocultures with antigen specific cytotoxic T cells and autologous tumor-infiltrating T cells. Death inducing intrinsic and extrinsic apoptotic pathways affected by TSPO were investigated. TSPO-regulated genes mediating apoptosis resistance in BTICs were identified through gene expression analysis and subsequent functional analyses. TSPO transcription in primary GB cells correlated with CD8+ T cell infiltration, cytotoxic activity of T cell infiltrate, expression of TNFR and IFNGR and with the activity of their downstream signalling pathways, as well as with the expression of TRAIL receptors. Coculture of BTICs with tumor reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through T cell derived TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO selectively protected BTICs against TRAIL-induced apoptosis by regulating apoptosis pathways. TSPO also regulated the expression of multiple genes associated with resistance against apoptosis. We conclude that TSPO expression in GB is induced through T cell-derived cytokines TNFα and IFNγ and that TSPO expression protects GB cells against cytotoxic T cell attack through TRAIL. Our data thereby provide an indication that therapeutic targeting of TSPO may be a suitable approach to sensitize GB to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL resistance