here number, similar to those tumorspheres treated with Compound X over both passages. Similar results were obtained with UI226 primary SHH MB cells that are nearly 100% for CD271 ( Figure  2A) After treatment with Compound X, we observed a significant increase in the total number of tumorspheres ( Figure 5A-5C) concomitant with a decrease in UI226 tumorsphere size relative to DMSO treated cells ( Figure   5A-5B,5D). We did not observe changes in cell viability; however, there was a significant decline in cell number in passage 1 spheres ( Figure 5E-5F). Secondary tumorsphere numbers were also significantly increased concomitant with a decreased tumorsphere size ( Figure 5G-5J). Similar to Daoy CD271 OE cells, secondary tumorspheres derived from cells treated with Compound X in passage 1 ("previously treated") also showed a decrease in tumorsphere size ( Figure 5J); however, in this case, the increased number of spheres was not sustained and was actually lower compared with DMSO controls ( Figure 5I). In addition, cell viability was not significantly different, while the total number of cells decreased ( Figure 5K-5L). For UI226 cells, Compound X treatment may need to be sustained over subsequent passage to continually affect all cell properties in tumorsphere culture. Collectively, these results demonstrate that blocking of CD271 processing by γ-secretase inhibitor reverses the OE phenotype observed in SHH MB cells.

CD271 knockdown results in generation of smaller tumorspheres in vitro
To complement our CD271 overexpression studies and to look at specificity of CD271 using a more direct method, we generated CD271 knockdown (KD) cells from our CD271 OE line as well as MED311-FH and UI226 SHH cells which, as opposed to Daoy, were originally derived and cultured in stem-cell propagating conditions and therefore express very high endogenous levels of CD271 (Figure 2A). Two different shRNA sequences targeting CD271 were used, along with a scrambled negative control. Both sequences resulted in a KD of CD271 compared to scrambled in all cells tested ( Figure 6A,6F,6K). Similar to the effects seen in our γ-secretase inhibitor treatment, CD271 KD in Daoy OE cells exhibited an increase in tumorsphere number and decrease in tumorsphere size compared to controls in P1 and P2 tumorspheres ( Figure 6B-6E). CD271 KD in MED311 and UI226 cells also results in a decrease in P1 and P2 tumorsphere size compared to controls ( Figure 6G-6J, 6L-6O); however, no significant changes in tumorsphere number were observed ( Figure 6I-6J, 6N-6O). We did not observe consistent changes in total cell counts and viability as measured by Trypan blue staining in Daoy OE ( Figure S4A-S4D), UI226 ( Figure S4E-S4H) or MED311 cells ( Figure S4I-L). Cell cycle gene (CDK2, CDK6, CCND1, CCND2) expression was also measured by qPCR in UI226 and MED311KD cells ( Figure S4M-S4N), and the differences were either not significant or inconsistent between the 2 shRNA sequences; however, there was a trend towards increased expression of CCND1 and CCND2 in UI226 cells. These KD data provide additional support for our overexpression studies and suggests that CD271 plays a role in regulating the SHH MB stem/progenitor cell state.

CD271 levels are inversely correlated with expression of SHH signaling pathway genes
Since we are utilizing a SHH MB variant for our studies and SHH pathway genes have been shown to be upregulated in CD133+ putative stem cell populations in Daoy cells [24], we examined expression of the SHH signaling pathway genes in CD271+ vs. CD271-cells and in CD271 OE vs. control cells by qPCR. We observed a significant downregulation of Patched (PTCH), Smoothened (SMO), GLI1, and GLI2 in Daoy sorted CD271+ compared to CD271-cells and a significant downregulation of GLI1 and GLI2 in MED311 sorted CD271+ vs. CD271-cells ( Figure 7A-7B). qPCR analysis of SHH pathway genes in CD271 OE cells compared to controls also revealed a downregulation of SMO, GLI1, and GLI2 (Figure7C). In contrast, following knockdown of CD271 in MED311 and UI226 cells, we typically observed an increase in expression of the downstream SHH pathway genes, GLI1 and GLI2, for both cell lines ( Figure 7D-7E). These results demonstrate that higher CD271 levels are negatively correlated with expression of SHH signaling pathway genes.

dIscussIon
In this study, we utilized an integrated approach of high throughput flow cytometry screening combined with gene expression profiling to identify cell surface markers that are differentially expressed in self-renewing vs. non-self-renewing SHH MB cells as well as in SHH MB vs. other MB variants. The Lyoplate platform has been utilized for characterizing specific cell phenotypes in multiple cancers [19,21]; however, this technique has not been used in pediatric cancers such as MB. While this study focused on characterization of CD271, our results also show that CD171 is a candidate for additional functional testing, as this cell surface marker has been linked with tumor progression, metastasis and therapeutic resistance in a wide variety of cancers [45][46][47][48][49][50][51][52][53].
We have demonstrated a novel role for CD271 in regulating the SHH MB stem/progenitor cell state and present a working model whereby CD271 is associated with a dynamic SHH MB progenitor or lower selfrenewing stem cells within a heterogeneous tumor population ( Figure 7F). CD271 is a transmembrane glycoprotein that plays a variety of roles in normal neurodevelopment including growth cone elongation, axon guidance, cell survival and cell death, depending on the cellular context [22]. The four CD271 ligands, NGF, BDNF, NT-3 and NT-4 exist in both pro-neurotrophin form and mature form, and this results in different effects depending on the presence/absence of ligand as well as which ligand is bound [22]. For example, CD271 has been shown to induce apoptosis in the absence of ligand or binding of an unprocessed neurotrophin (pro-neurotrophin) [54]. Activation of the JNK pathway appears to play a role in CD271 mediated cell death, whereas signaling through the NFκB pathway promotes survival [54]. Future studies will determine which pathways are regulated by CD271 in SHH MB progenitors and stem cells. In addition to its role in neurodevelopment, CD271 has been shown to regulate adult glioblastoma brain tumor invasion and has been shown to be a selective tumor propagating cell marker in melanoma, esophageal squamous cell carcinoma, and hypopharyngeal cancer [42,55]. Importantly, CD271 has also been shown to mark a neurogenic precursor population, which includes stem and progenitor cells, in the subventricular zone (SVZ) from both rats and mice [56]. Using FACS to isolate CD271+ postnatal rat SVZ, the authors showed that sorted cells with the highest levels of CD271 generated the most neurospheres. They also demonstrated that CD271 regulates neurogenesis and the ongoing generation of olfactory bulb neurons in the SVZ [56].
Similarly, our results suggest that CD271 selects for a stem/progenitor cell population in tumorsphere assays. However, there is conflicting evidence in the literature regarding the interpretation of sphere size changes and multiple explanations have been proposed [57]. For example, larger spheres have been positively correlated with the number of stem cells and capacity for self-renewal. However it is also possible that size is merely related to differentiation potential, proliferation of progenitor cells and/or response to growth factors [57]. Thus, larger spheres may actually contain more progenitors as opposed to stem cells. In our model systems, we did not observe any consistent changes in proliferation; however, future studies will evaluate the effect of CD271 on differentiation of SHH MB cells in the presence of its ligands. Given the difficulties in interpreting sphere size data, we have not distinguished between a stem or progenitor cell in our gain/loss of function studies. CD271 was typically associated with an overall lower self-renewal capacity but an increased sphere size.
Studies conducted in mouse models have shown that the SHH variant of MB arises from granule precursor cells in the external granular layer (EGL) of the cerebellum [39,40]. In addition, Li et al. showed that a population of nestin-expressing progenitor cells, distinct from granular neuroprogenitor cells in the EGL, are responsible for tumorigenesis [58]. CD271 immunostaining in paraffin embedded sections of 23-week human fetal cerebellum ( Figure 2B) revealed high expression of CD271 in the EGL, an area with a high density of progenitor cells, and is consistent with staining profiles previously observed [59,60], In MB, previous work has demonstrated conflicting roles for CD271 in MB apoptosis [61,62]; however, in both cases, experiments were conducted with D283, or non-SHH MB cells, further underscoring the need to evaluate the function of a cell surface marker in both a subtype-specific and cell context-dependent manner.
Activation of the SHH signaling pathway is characteristic of SHH MB. Studies utilizing SHH pathway inhibitors have shown that patients display an initial response, however this is followed by eventual relapse attributed to drug resistance [63][64][65][66]. Zhukova et al. recently shed light on this issue by demonstrating that P53 mutations