Abstract
Medulloblastoma (MB) is the most common malignant primary brain tumor and is currently classified into 5 distinct molecular subtypes based on genomic alterations, gene expression profile, response to treatment and cell of origin. This extensive heterogeneity has revealed a critical need for subtype-specific, functionally validated biomarkers and therapeutic strategies. Using an unbiased high throughput flow cytometry screen and gain/loss of function studies, we previously identified CD271/p75NTR as a candidate stem/progenitor cell marker, specifically in SHH MB. Here, we show that CD271+ and CD271- subpopulations do not exhibit a hierarchal organization; but are rather two co-existing cellular subsets. FACS sorting followed by re-culturing of CD271- and CD271+ cells from low passage primary cultures demonstrated that both cell populations can recapitulate the parental phenotype. However, global gene expression profiling by RNA sequencing revealed that genes/pathways associated with cell survival, proliferation and motility are downregulated in CD271- vs. CD271+ subpopulations. Cell cycle analysis supported these findings and demonstrated that CD271- cells exhibited a decrease in proliferation compared to CD271+ cells. Moreover, PI3K/AKT, JAK/STAT, and NFkB, are downregulated in CD271- vs. CD271+ cells, while pathways such as RAS/MYC are upregulated in CD271- vs. CD271+ cells. Immunohistochemical (IHC) analysis of CD271 levels across the MB subtypes demonstrated that nearly all SHH tumors express CD271, while Group 3 tumors exhibit moderate staining. In contrast, Group 4 MB and WNT do not express CD271. Importantly SHH MB exhibit a nodular CD271 staining pattern, further underscoring the heterogeneity within these tumors and supporting our findings that CD271- and CD271+ cells are distinct, yet clinically relevant, subpopulations. Our results suggest that CD271, in combination with other markers, could be effectively utilized as a diagnostic tool for SHH MB and that therapeutic strategies concomitantly targeting both CD271+ and CD271- subpopulations would be the most effective in SHH MB treatment.