108P - Establishing a multi-modal tissue preparation and imaging workflow to study heterogeneity in neuroblastoma tumors

Background

Neuroblastoma (NB) is the most common solid tumor in infants and accounts for 15 % of childhood related cancer deaths. Clinical, genetic and phenotypic heterogeneity of NB hamper patient stratification, challenging therapeutic approaches. Methods to investigate tumor cell phenotypes and cells of the microenvironment in the primary tumors have so far mostly focused on transcriptomics or have been limited to only a few phenotypic markers on the protein and tissue level.

Methods

Multiplexed imaging techniques allow simultaneous or sequential detection of a high number of phenotypic and genetic markers. We aimed to establish a multi-modal tissue preparation and imaging workflow to integrate these multiplexed imaging techniques and thereby increase the confidence in tumor cell identification while studying tumor heterogeneity in NB at a high-dimensional scale. We combined three different imaging methods, i.e., 3-plex immuno-fluorescence staining (IF), 3-plex interphase fluorescence-in-situ-hybridization (iFISH) and 42-plex imaging mass cytometry (IMC).

Results

The multi-modal imaging method was established on NB tumor sections. First, all imaging techniques were optimized separately and subsequently in a combined fashion to generate sample preparation and staining protocols for combined IF and iFISH as well as for combined IF and IMC. Once the workflow was set, we assembled a patient cohort of 34 fresh frozen, high-risk NB tumors for analysis. For each sample we prepared two consecutive 4 μm thick cryosections, which were IF-stained according to the protocol to identify regions of interest for IMC and to align both tumor slices. Upon IF, one section was used for IMC to investigate the presence of different tumor cell phenotypes and immune infiltrates, while the other slice was used for iFISH to detect patient specific genetic aberrations.

Conclusions

The proposed workflow enables the analysis of the same tissue region with different imaging modalities, which would otherwise not be possible due to destructive properties or chemical incompatibilities of standard imaging protocols. This workflow will be of great benefit to reveal tumor heterogeneity and spatial relations of tumor cells with their microenvironment in NB.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

S. Taschner-Mandl.

Funding

Austrian Science Fund (FWF).

Disclosure

All authors have declared no conflicts of interest.