Deciphering the mechanisms of neuroblastoma metastasis by multi-omics analysis

[eng] Neuroblastoma (NB), a heterogeneous pediatric cancer originating from neural crest cells, presents significant clinical challenges due to its high propensity for metastasis. Approximately 60% of patients show widespread disease at the initial diagnosis, with metastasis being the primary cause of NB-related deaths. Understanding the molecular mechanisms underlying metastasis is crucial for the development of targeted therapies for patients with high-risk NB. The metastatic process involves complex interactions between tumor cells and their surroundings, with the tumor microenvironment (TME) playing a key role. Current in vivo models for NB metastasis have limitations, including incomplete replication of metastatic stages and difficulties in tumor tracking and removal. To address these issues, we established a spontaneous metastasis in vivo model, allowing surgical removal of the primary tumor, ensuring animal survival, and enabling metastatic cells to colonize distant organs. Our primary objective was to identify a metastatic signature by multi-omics analyses, comparing primary tumors and metastatic masses from the in vivo model. Whole-genome expression analysis revealed that the most differentially expressed genes (DEGs) were linked to lymphatic metastasis. Data integration emphasized the crucial role of the tumor microenvironment (TME) in NB metastasis, particularly the TGF-β signaling pathway, suggesting a potential interaction between TME and NB mesenchymal (MSN) phenotype induction. Podoplanin (PDPN), a transmembrane glycoprotein, has emerged as a metastatic marker for NB. It exhibited distinct cellular localization in NB cells, with a prominent cytoplasmic location in metastasis. Notably, the membrane localization of PDPN in NB cells was linked to higher lymph node infiltration rates in NB patients. By generating NB stable cell lines overexpressing PDPN, we demonstrated that elevated levels were correlated with increased metastatic potential. Since PDPN expression is known to be induced by interactions with stromal and immune cells, we confirmed that NB cells can promote the polarization of fibroblasts into cancer-associated fibroblasts (CAFs) and that PDPN is induced in NB cells through interactions with CAFs, suggesting a potential feedback loop. Furthermore, PDPN expression was also induced by low oxygen conditions and cytokines exposure, confirming significant regulation by TME. Notably, NB cells released PDPN via exosomes to facilitate the establishment of premetastatic niches by inducing stromal cell polarization. Altogether, our data provide further insight into the mechanisms underlying metastasis in NB and identify a novel NB metastatic driver to target, crucial to improve patients' outcome.