Characterization of cellular heterogeneity in the SHH subgroup of medulloblastoma

Abstract

Medulloblastoma (MB) is the most common form of primary malignant pediatric brain cancer. MB is divided into 5 molecular subgroups; Wnt, Sonic Hedgehog (SHH) p53 mutant, SHH p53 wildtype, Group 3 and Group 4. Major research efforts have focused on the isolation and characterization of MB brain tumor stem cells, also known as brain tumor propagating cells (BTPC). Elucidating cell surface marker profiles that can be used to selectively isolate this cellular population is an imperative first step in the development of targeted therapies. Given the variable results obtained for currently utilized markers, as well as the cellular heterogeneity within and between MB sub-groups, it is likely there are additional surface marker profiles capable of selecting for sub-type specific MB BTPCs. We set out to identify novel surface marker combinations capable of selecting for BTPCs in SHH MB. We employed the BD Bioscience Lyoplate screening platform to compare the levels of 242 human cell surface markers in high and low self-renewing SHH MB sub-clones. The top 25 markers showing the greatest differences were refined by evaluating expression levels in SHH vs Group 3, Group 4 and Wnt variants in transcriptome datasets representing 548 patient samples. Four markers, CD271, CD106/VCAM1, EGFR and CD171/NCAM-L1 showed consistent differential expression in the SHH subtype relative to the other variants. Flow cytometry validation in additional cell lines as well as IHC in patient samples confirmed these findings. Our laboratory focused on the functional validation of CD271 and CD106 and the role they play in MB tumorigenesis. Functional validation of CD271 in SHH MB in vitro and in vivo was performed. Using sorted cell populations and gain/loss of function studies, we showed that CD271 is a subtype specific marker that selects for a cellular population in the progenitor/stem cell state in SHH MB. Initial functional characterization of CD106 showed no effect of this cell surface marker on invasion, self-renewal or proliferation in vitro. Additional studies evaluated the generally accepted principle that cell populations with higher self-renewal capacity in vitro generate larger tumors and exhibit increased tumor penetrance as well as decreased survival in vivo. We demonstrated that a SHH MB subclone derived from the Daoy cell line with a lower self-renewal capacity in vitro, when injected into the frontal cortex of NOD SCID mice, result in a shorter survival and increased tumor grade, when compared to a subclone derived from the same Daoy cell line displaying a higher self-renewal capacity. These results challenge the prevailing notion that results in vitro vs. in vivo are positively correlated. This thesis was able to show that SHH medulloblastoma is a heterogeneous subgroup that has subgroup specific cancer stem cell markers. We were able to show that CD271 is a SHH MB specific marker that plays a significant role in tumorigenic potential. Future research studying the eradication of this population holds a great deal of promise in treating medulloblastoma clinically.