Supplemental Table and Figure Legends
Supplemental Table S1. Hirsch Score was calculated from tumors and their respective stroma from Figure 5. Percentages represent the total staining (0, 1+, 2+, 3+) from the whole slide examined at 10x magnification. EGFR, Gli1, ALDH1, BMI1 were evaluated for all tumor samples and only Gli1 was evaluated for stroma.
Supplemental Table S2. All potential flank implanted stromal:tumor and tumor:tumor combinations were measured bi-weekly to chart growth kinetics. Fixed tumor measurement were performed at 3 weeks and 6 weeks. Tumors that did not grow were still counted in calculating overall mean, SD and SEM.
Supplemental Figure S1. Comparison of HN11 and TU167 by RNA-Seq Expression. Total mRNA from HN11 and TU167 were submitted for analysis. Clusters of gene pathways were evaluated between the two cell lines for differences in expression. Pathways evaluated included Hedgehog, Epithelial Cell Signaling and mTOR. Samples were submitted in duplicate. P-values <0.05 were considered significant.
Supplemental Figure S2. Suppression of Radiation-induced GLI1 using shRNA Model. HN11 and TU167 shScramble and shGLI1 cell lines grown in the presence of doxycycline were irradiated. GLI1 gene expression was evaluated by qRT-PCR. Statistically significant findings were denoted: *P<0.05; **P<0.01; ***P< 0.001.
Supplemental Figure S3. Cyst Formation Following Radiation Treatment in Orthotopic TU167-implanted Tumors. A. Cystic (Left) and non-cystic (Right) orthotopic tumors following 3 weeks of dual therapy. B. RT and dual therapy treated tumors (cystic and non-cystic lesions) were resected at time of euthanasia. Cystic and non-cystic lesion were evaluated by IHC (5x, bar 100M) for H/E, EGFR, Ki67, E-cadherin and CD31. E-cadherin and CD31 not shown. C. IHC (10x, 100M) of resected in vivo TU167 tumors stained for Ki67.
Supplemental Figure S4. Radiation-induced Gli1 Expression and the Effect of Rapamycin Inhibition in HN11 Cells. Nuclear and cytoplasmic extracts were generated as previous described from HN11 treated with ±1M rapamycin ±RT. cGli1 and nGli1 accumulation was evaluated by immunoblot and temporal fold change were described.
Supplemental Figure S5. Effect of siRNA inhibition on Head Neck Cancer Cells and Gli1 Nuclear Translocation. HN11 (Top) and TU167 (Bottom) was transiently transfected with a scramble construct and two S6K1 siRNAs. Following transfection, cells were irradiated 48 hours after transfection and protein harvested 4 hours after RT for immunoblot.
Supplemental Figure S6. Schema and Histology Detailing Radiation-induced Tumor Repopulation. A. Schema detailing pretreatment of TU167 FOTM tumors, tumor harvest, flow sorting into mouse tumor stroma and human tumor cell components and re-implantation into the flanks of new athymic nude mice. 5000 treated (Control, Cyclopamine, RT or Dual) stroma or tumor cells were mixed with 15,000 fresh, untreated TU167 cells; stroma:tumor mixed cells were implanted on the left flank and tumor:tumor mixed cells were implanted on the right flank. Tumors were measured bi-weekly for tumor growth and animals euthanized and tumors harvested at 6 weeks post-implantation. Stroma-derived from the control, cyclopamine, RT or dual treatment arm, respectively, and mixed with fresh tumor were denoted as SC:T, SCyc:T, SRT:T, SD:T. Tumor cells derived from the control, cyclopamine, RT or dual treatment arm, respectively, and mixed with fresh tumor were denoted as TC:T, TCyc:T, SRT:T, SD:T. B and C. Tumors were harvested at 6 weeks and H/E was performed on tumors from each of the 4 treatment arms and either from group S:T (B) versus T:T (C). Images were captured at 10X magnification.