Rising litigation does not mean falling standards
Authors: P. Nisselle
Journal: BMJ (2012)

<h3>Background</h3> CAR-T cell-based therapies hold great promise for neuroblastoma, because of its potential for targeted effects and already promising results in adult solid cancers. However, neuroblastoma tumour cells can escape T cell mediated killing by inhibition of T cells through secretion of immunosuppressive factors. Here, we have identified Macrophage Migration Inhibitory Factor (MIF) and Midkine (MDK) by scRNA-seq analysis to have a negative effect on the cytotoxicity of T cells. Next to that, we have found both MIF and MDK in high abundance in the secretome of neuroblastoma patient derived organoids. These factors have a suppressive effect on T cells and blocking their function might increase the efficacy of T cell based therapies for neuroblastoma. <h3>Materials and Methods</h3> To study immunoregulatory interactions in neuroblastoma, single-cell RNA-sequencing of 25 tumours were analysed using the CEL-seq2 platform. Interactions between tumour and immune cells were predicted using an unbiased ligand-receptor interaction analysis. Proteins secreted by tumour cells were analysed by performing mass spectrometry on conditioned medium from patient derived organoid cultures. The conditioned medium was concentrated using a 3kDa Millipore filter and prepared for LC-MS. Mass spectrometry data were acquired in data-dependent acquisition mode. For determining suppression, healthy donor PBMCs were stimulated with anti-CD28/anti-CD3 Dynabeads in the presence of recombinant proteins or concentrated conditioned medium from neuroblastoma organoids. In vitro killing assays were performed with GFP and luciferase transduced organoids and healthy donor PBMCs. <h3>Results</h3> Analysis of scRNA-seq of 25 neuroblastoma tumours showed a negative correlation between MIF and MDK expression and the cytotoxicity of NK cells, CD8 and γδ-T cells within the tumour. These findings could be confirmed with a previously published SEQC bulk-RNAseq dataset containing 498 patients. Next to that, a higher expression of MIF and MDK correlated with poor survival in the same dataset. In the secretome from cultured neuroblastoma organoids, we have used mass spectrometry to identify MIF and MDK amongst the top 100 most abundant proteins from a total of ~1200 proteins. In vitro, we showed that rMIF and rMDK have a suppressive effect on the activation of T cells and the amount of cytotoxic factors such as granzyme B and Perforin are produced by the T cells. This confirms our hypothesis that MIF and MDK negatively influence the cytotoxicity of T cells. <h3>Conclusions</h3> Using two different unbiased analyses -scRNA-seq analysis of tumours and mass spectrometry of neuroblastoma organoid secretome-, we have identified MIF and MDK as immunosuppressive factor in neuroblastoma. Currently, we are testing several MIF and MDK inhibitors to test if T cell mediated killing of neuroblastoma can be increased if the immunosuppressive MIF and MDK are blocked. <h3>Disclosure Information</h3> <b>J.G.M. Strijker:</b> None. <b>R.E. van Berkum:</b> None. <b>M. Damen:</b> None. <b>W. Wu:</b> None. <b>J.J. Molenaar:</b> None. <b>J. Wienke:</b> None.