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

Abstract

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-naïve MB. Though astrocytes have been frequently detected in brain tumors, their roles in regulating the stemness 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 stemness, 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 necroptotic TAA. 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 stemness maintenance in disseminations. Key Points 1. TAA-derived CCL2 promoted stemness in disseminated MBSCs through Notch signaling activation via the JAK2/STAT3 pathway. 2. TAA released CCL2 in a RIP1/RIP3/MLKL-dependent manner leading to necroptosis.

Astrocytes influence medulloblastoma phenotypes and CD133 surface expression

“…It is known that Daoy MB cells cultured as neurospheres express higher CD133 (a pentaspanin transmembrane glycoprotein that has been associated with protrusion formation and localization [26]) when compared to Daoy cells cultured in an adherent monolayer [27,28]. Further, these neurosphere-forming MB cells were more invasive in vitro [27], and CD133 expression is high in disseminated human MB tumors [9]. Thus, we investigated CD133 surface expression in ACM-conditioned MB cells compared to control-media conditioned MB cells using flow cytometry.…”

“…The astrocyte secretome is diverse and thus, there are likely a combination of factors in the ACM influencing the MB phenotypes. CCL2, SHH, and IL-4 are astrocyte-secreted factors which have been shown to promote MB phenotypes [8,9,42]. Interestingly, CCL2 secretion from astrocytes [9] was induced by chemotherapy and radiation-induced inflammation and necroptosis in astrocytes.…”

“…CCL2, SHH, and IL-4 are astrocyte-secreted factors which have been shown to promote MB phenotypes [8,9,42]. Interestingly, CCL2 secretion from astrocytes [9] was induced by chemotherapy and radiation-induced inflammation and necroptosis in astrocytes. Moreover, the IL-4 secretion was found to come from astrocytes which trans-differentiated from the MB precursor cells in a SHH MB mouse model [42].…”

“…In primary brain tumors, such as MB, tumor-associated astrocytes have been found to secrete sonic hedgehog (SHH), which directly increased Nestin expression and proliferation of MB cells derived from a genetic mouse model of SHH MB [8]. Metastatic MB tumor-associated astrocytes were also recently identified to secrete chemokine C-C ligand 2 (CCL2), which enriched stem cell properties in MB cells, including expression of CD133 [9]. CD133 expression was also found to play a role in glioblastoma stem cells, wherein only CD133 positive cells showed increased invasion and radioresistance upon co-culture with astrocytes [10,11].…”

“…Here, CD133 positive cells were tumor initiating and grew as neurospheres in vitro, whereas CD133 negative cells lacked those properties [12]. Subsequent research on CD133's function in MB has shown that it plays a role in cell survival and therapy resistance through several pathways, including PI3K/AKT [13,14], Notch [15], and STAT3 [9,16]. Thus, interest in CD133 as both a target and a regulatory molecule in the MB tumor microenvironment is high.…”