Necroptotic astrocytes contribute to maintaining stemness of disseminated medulloblastoma through CCL2 secretion

Background. Medulloblastoma (MB) with metastases at diagnosis and recurrence correlates with poor prognosis. Unfortunately, the molecular mechanism underlying metastases growth has received less attention than primary therapy-naive MB. Though astrocytes have been frequently detected in brain tumors, their roles in regulating the sternness properties of MB stem-like cells (MBSCs) in disseminated lesions remain elusive. Methods. Effects of tumor-associated astrocyte (TAA)-secreted chemokine C-C ligand 2 (CCL2) on MBSC self-renewal was determined by immunostaining analysis. Necroptosis of TAA was examined by measuring necrosome activity. Alterations in Notch signaling were examined after inhibition of CCL2. Progression of MBSC-derived tumors was evaluated after pharmaceutical blockage of necroptosis. Results. TAA, as the essential components of disseminated tumor, produced high levels of CCL2 to shape the inflammation microenvironment, which stimulated the enrichment of MBSCs in disseminated MB. In particular, CCL2 played a pivotal role in maintaining stem-like properties via Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3)-mediated activation of Notch signaling. Loss of CCL2/C-C chemokine receptor 2 (CCR2) function repressed the JAK2/STAT3-Notch pathway and impaired MBSC proliferation, leading to a dramatic reduction of sternness, tumorigenicity, and metastasizing capability. Furthermore, necroptosis-induced CCL2 release depended on activation of receptor-interacting protein 1 (RIP1)/RIP3/mixed lineage kinase domain-like pseudokinase (MLKL) in TAA, which promoted the oncogenic phenotype. Blockade of necroptosis resulted in CCL2 deprivation and compromised MBSC self-proliferation, indicating MBSCs outsourced CCL2 from necroptoticTAA. Finally, CCL2 was upregulated in high-risk stages of MB, further supporting its value as a prognostic indicator. Conclusion. These findings highlighted the critical role of CCL2/CCR2 in Notch signaling activation in MBSCs and revealed a necroptosis-associated glial cytokine microenvironment driving sternness maintenance in disseminations.