Background The development of immunotherapeutic agents has transformed the treatment of neuroblastoma patients. Nevertheless, the efficacy of these immunotherapies is challenged by the highly immunosuppressive microenvironment, which hampers immune cell migration into tumor area. Here, we aim to identify targeted strategies to enhance immune cell infiltration, by mapping the tumor immune microenvironment (TIME) and cell-cell interactions in neuroblastoma using imaging mass cytometry (IMC).

Materials and Methods We used IMC to profile the in situ expression of 36 proteins in paraffin-embedded sections of resected neuroblastoma tumors of 14 high risk patients (6 MYCN-amplified and 8 MYCN non-amplified) after induction chemotherapy, including two matched relapse samples. Our lymphocyte-focused antibody panel consisted of multiple lineage markers to distinguish distinct (immune) cell subsets, in addition to checkpoint, exhaustion and cytotoxicity markers. We performed distance and nearest neighbor analyses to determine the different cell-cell interactions in combination with their spatial distribution in the tissue.

Results Neuroblastoma revealed a heterogeneous immune environment primarily composed of macrophages and CD4+ as well as CD8+ T cells. Additionally, we identified γδ T cells and B cells, whereas NK cells were limited. Interestingly, we observed three different spatial patterns of T cell clusters: (1) in close proximity of blood vessels, (2) in the stroma and or tumor margin, and (3) infiltrated in the tumor area. Further analysis on the expression of specific activation, cytotoxicity and/or exhaustion markers such as Granzyme B, CD137 and TOX is currently ongoing to provide more insight in the functional differences between T cells in these three areas. Furthermore, we observed that most macrophages were localized in close proximity of T cells suggesting a possible inhibitory interaction. In addition, we identified the expression of inhibitory receptors like TIGIT and PD-1 on T cells, which further reveals the potential immunosuppressive mechanisms in neuroblastoma. Moreover, by combining all these data with clinical information such as MYCN-status and response to chemotherapy, we aim to identify specific immune infiltration patterns across these groups.

Conclusions With this study we reveal the spatial network of immune subsets and their interactions inside neuroblastoma tumors. These insights will be at the base for the development of novel and more personalized immunotherapeutic strategies to increase the survival rate of patients with high-risk neuroblastoma.

F.J. Bergsma: None. D. Krijgsman: None. F. van den Ham: None. M. Amini: None. M. Verdonschot: None. S. van Dam: None. R. de Krijger: None. J. Molenaar: None. Y. Vercoulen: None. J. Wienke: None.