Supplementary Data from iNOS Regulates the Therapeutic Response of Pancreatic Cancer Cells to Radiotherapy
Authors: Pereira Patricia M.R. , Edwards Kimberly J. , Mandleywala Komal , Carter Lukas M. , Escorcia Freddy E. , Campesato Luis Felipe , Cornejo Mike , Abma Lolkje , Mohsen Abu-Akeel , Iacobuzio-Donahue Christine A. , Merghoub Taha , Lewis Jason S. 
Published: April 2023

<p>Supplementary Methods Supplementary Figure 1. iNOS staining in non-tumor and PDAC tumors obtained before treatment and after RT. Supplementary Figure 2. Western blot of iNOS in the total lysates of FC1245 orthotopic PDAC tumors obtained 2 days post-RT therapy. Supplementary Figure 3. Immunohistochemistry of trichrome in FC1245 orthotopic PDAC tumors obtained at 2 days post-RT therapy. Supplementary Figure 4. CAFs isolation and expansion from FC1245 orthotopic PDAC tumors. Supplementary Figure 5. Timeline for FC1245 culture with CM from CAFs or co-cultured with CAFs. Supplementary Figure 6. Cell growth of FC1245 cultured in the presence or absence of conditioned media from irradiated CAFs or co-cultured with irradiated CAFs. Supplementary Figure 7. NO2- levels in supernatants of human pancreatic cancer cell lines in the presence and absence of supernatants from human CAFs and treated with iNOS inhibitor 1400W. Supplementary Figure 8. IL-6, LIF, RANTES, and IL-13 concentrations in the supernatants of FC1245 PDAC cells, FC1245 incubated with conditioned medium from non-RT CAFs, and FC1245 incubated with conditioned medium from RT CAFs. Supplementary Figure 9. Gene expression heatmap of non-RT and RT fibroblasts. Supplementary Figure 10. Confocal images and quantification (meanâ€‰{plus minus}â€‰S.E.M, nâ€‰=â€‰3) of immunofluorescence staining of Î±-SMA (green) in FC1245 orthotopic PDAC tumors at 14 days after cells' implantation. Supplementary Figure 11. FC1245 migration images and quantification using the wound healing assay. Supplementary Figure 12. Liver and pancreatic tumors at 6 days after surgery. Supplementary Figure 13. Liver, stomach, spleen, and pancreatic tumors at 14 days after surgery. Supplementary Figure 14. FC1245 cell growth and NO2- levels in supernatants of FC1245 with or without DETA-NONOate, 1400W or with the combination of DETA-NONOate/1400W. Supplementary Figure 15. FC1245 cell growth and NO2- levels in supernatants of FC1245 transfected with iNOS CRISPR activation plasmid or control CRISPR activation plasmid. Supplementary Figure 16. Cell growth of FC1245 cultured in the presence or absence of irradiated and non-irradiated CAFs. Supplementary Figure 17. Survival of mice implanted with FC1245 orthotopic PDAC tumors and treated with RT. Supplementary Figure 18. Tumor weight in PDAC tumors from RT, 1400W, and 1400W/RT treated mice on day 14 post-therapy. Supplementary Figure 19. Glucose uptake and lactate production of FC1245 PDAC cells treated with or without DETA-NO, 1400W, and the combination of DETA-NO/1400W. Supplementary Figure 20. Biodistribution data at 2 h after injection of 18F-FDG in mice bearing FC1245 orthotopic tumors. Supplementary Figure 21. Biodistribution data at 24 h after injection of 67Ga-labeled pHLIP in mice bearing FC1245 orthotopic tumors. Supplementary Table 1. RT administrations, time-points, and human PDAC sample collection time-points. Supplementary Table 2. Quantification of iNOS+/Î±-SMA+ in PDAC RT-treated samples obtained from patient #3. Supplementary Table 3. Quantification of iNOS+/cytokeratin+ in PDAC RT-treated samples obtained from patient #3. Supplementary Table 4. Quantification of iNOS+/Î±-SMA+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Supplementary Table 5. Quantification of iNOS+/Î±-SMA+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Supplementary Table 6. Quantification of iNOS+/cytokeratin+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Supplementary Table 7. Quantification of iNOS+/cytokeratin+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Supplementary Table 8. Quantification of GLUT1+/Î±-SMA+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Supplementary Table 9. Quantification of GLUT1+/Î±-SMA+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Supplementary Table 10. Quantification of GLUT1+/cytokeratin+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Supplementary Table 11. Quantification of GLUT1+/cytokeratin+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors.</p>