Cryptic developmental events determine medulloblastoma radiosensitivity and cellular heterogeneity without altering transcriptomic profile.

Abstract

Patients with medulloblastoma are typically treated with a narrow range of therapies, but may experience widely divergent outcomes; 80-90% become long-term survivors while 20% develop incurable recurrence. Transcriptomic profiling has identified four subgroups with different recurrence risks, but outcomes remain variable for individual patients within each subgroup. To gain new insight into why patients with similar-appearing tumors have variable outcomes, we examined how the timing of tumor initiation effects medulloblastomas triggered by a single, common driver mutation. We genetically-engineered mice to express an oncogenic Smo allele starting early in development in the broad lineage of neural stem cells, or later, in the more committed lineage of cerebellar granule neuron progenitors. Both groups developed medulloblastomas and no other tumors. We compared medulloblastoma progression, response to therapy, gene expression profile and cellular heterogeneity, determined by single cell transcriptomic analysis (scRNA-seq). The average transcriptomic profiles of the tumors were similar. However, stem cell-triggered medulloblastomas progressed faster, contained more OLIG2-expressing tumor stem cells, and consistently showed radioresistance. In contrast, progenitor-triggered MBs progressed slower, lost stem cell character over time and were radiosensitive. Progenitor-triggered medulloblastomas also contained more diverse stromal populations, including tumor-associated macrophages, indicating that the timing of oncogenesis affected the subsequent interactions between the tumor and microenvironment. Our findings show that developmental events in tumorigenesis may be impossible to infer from transcriptomic profile, but while remaining cryptic can nevertheless influence tumor composition and the outcome of therapy. Precise understanding of medulloblastoma pathogenesis and prognosis requires supplementing transcriptomic data with biomarkers of cellular heterogeneity.

Enhancing CDK4/6 inhibitor therapy for medulloblastoma using nanoparticle delivery and scRNA-seq-guided combination with sapanisertib

“…The resulting G-Smo mice developed medulloblastoma with 100% penetrance by P10 and, untreated, died of progressive tumors by P20, as in prior studies (16)(17)(18)(19). Consistent with the previously reported data, oral dosing or systemic dosing of free palbociclib (Palbo-HCl) didn't induce survival benefits.…”

“…We generated mice with SHH medulloblastoma by breeding hGFAP-Cre mice that express Cre reombinase in CNS stem cells during development with SmoM2 mice that express a Cre-conditional oncogenic allele of Smo . The resulting G-Smo mice developed medulloblastoma with 100% penetrance by P10 and, untreated, died of progressive tumors by P20, as in prior studies ( 17 , 18 ). We determined the maximum tolerated doses (MTD) for healthy WT mouse pups treated daily with oral Palbo-HCl starting at P10, or parenteral Palbo-HCl, administered IP daily from P10-P14, then every other day (Fig.…”

“…We used Drop-seq ( 22 ) methods as in our prior studies ( 21 , 23 ) to compare tumors isolated from three P15 G-Smo mice treated with 25 mg/kg POx-Palbo daily IP for 5 days to tumors from five age-matched, untreated G-Smo mice. Briefly, tumors were harvested, dissociated and individual cells were paired with bar-coded, oligo dT-coated beads using microfluidic methods, and bar-coded cDNA was synthesized on the beads.…”

“…As in our prior medulloblastoma scRNA-seq studies, the UMAP showed both discrete clusters and a set of clusters with shared borders. We identified each cluster using markers of proliferation and CGNP lineage to identify tumor cells, and using markers of different types of stromal cells expected in the brain, as validated in our prior medulloblastoma scRNA-seq studies ( 21 , 23 ). These markers showed that each discrete cluster comprised a different type of stromal cell including astrocytes, oligodendrocytes, myeloid cells, endothelial cells and fibroblasts ( Fig.…”

“…As in our prior medulloblastoma scRNA-seq studies, cells grouped in either discrete clusters or in a set of clusters with shared borders. We identified each cluster using stromal cell markers and the CGNP lineage marker, as validated in our prior medulloblastoma scRNA-seq studies (17,22). These markers showed that each discrete cluster comprised a different type of stromal cells typical of brain tissue including astrocytes, oligodendrocytes, myeloid cells, endothelial cells and fibroblasts and the 6 clusters in the multicluster complex as medulloblastoma cells (Fig.…”