Satoshi Nakano (Nagoya / JP; Freiburg / DE), Konrad Kurowski (Freiburg / DE), Ebrahem Hamed (Freiburg / DE), Hizuru Amano (Nagoya / JP), Akinari Hinoki (Nagoya / JP), Peter Bronsert (Freiburg / DE), Hiroo Uchida (Nagoya / JP), Oliver Schilling (Freiburg / DE), Simone Hettmer (Freiburg / DE; Halle (Saale) / DE)
Introduction:
Rhabdomyosarcoma (RMS) is the most common soft tissue tumor in childhood and adolescence. In PAX3::FOXO1 (P3F)-positive RMS, cell-to cell heterogeneity in fusion oncoprotein expression appears to play a crucial role in tumor progression and facilitate drug resistance. This study aims to elucidate intratumoral heterogeneity via Mass spectrometry (MS)-based proteomics.
Methods:
41 xenograft tumors – established from 6 different, low-passage patient-derived cell cultures - were harvested. Two peripheral tissue cores and one central core sample were collected from each tumor for a total of 123 samples. These samples were lysed with 1% SDS, followed by double digestion with Trypsin and Lys-C via the single-pot, solid-phase-enhanced sample-preparation (Sp3) protocol. MS-based proteomics were processed using Evosep One chromatography and timsTOF flex with long gradient and data independent acquisition (DIA)- Parallel Accumulation Serial Fragmentation (PASEF). Data analysis was performed using DIA-NN and R software.
Results:
Overall, 7235 unique human and murine proteins were detected. Following sparsity reduction, 4339 human (tumor) proteins and 1365 murine (stromal) proteins were further analyzed. The proteomic profiles of peripheral and core samples were similar. As expected, no differences were detected among murine proteins. For human (tumoral) proteins, the proteins belonging to plasma membrane and coated membrane pathways were significantly upregulated in peripheral regions, whereas the chromosome and centromeric region pathways were enriched in core regions.
Conclusions:
MS-based proteomics was used to investigate intratumoral heterogeneity of protein expression in P3F positive RMS. Mixed-species proteomics of a PDX model provided new insight into the tumor stroma components. Our future studies will validate differences in protein expression and explore the biological impact of such differences.