Figure S1 from Olaparib and Radiotherapy Induce Type I Interferon– and CD8⁺ T Cell–Dependent Sensitization to Immunotherapy in Pancreatic Cancer

Supplemental Figure 1. (A-S) Pancreatic cancer cells were treated with olaparib (5 µmol/L) one hour before radiation treatment (8 Gy) and collected 72 hours after treatment for analysis via flow cytometry, qRT-PCR or ELISA, unless otherwise denoted. (A) Panc-1 cells stably expressing IFNβ1-promoter GFP-reporter were treated with radiation and/or talazoparib (10 nmol/L or 30 nmol/L). Analyzed via flow cytometry for mean fluorescence intensity (MFI) of GFP. Data are the mean of 3 independent experiments. (B) KPC2 cells were treated with olaparib (3µmol/L or 5µmol/L) and/or radiation (8 Gy) and collected at 24 hours, 48 hours, and 72 hours after radiation treatment. qRT-PCR was conducted for Ifnβ. Data are the mean fold change ± SEM relative to control treatment for each time point and represent 1 independent experiment performed in technical triplicate. (C) Panc-1 cells in which cGAS was knocked out using a CRISPR/Cas9 system were treated with olaparib and/or radiation and harvested for analysis via western blot for pTBK1, pSTAT1, STAT1 and Actin. (D-F) KPC2 cells were treated with olaparib (3µmol/L or 5µmol/L) and/or radiation (8 Gy) and collected at 24 hours, 48 hours, and 72 hours after radiation treatment. qRT-PCR was conducted for Cxcl9 (D), Cxcl10 (E), and Mx1 (F). Data are the mean fold change ± SEM relative to control treatment for each time point and represent 1 independent experiment performed in technical triplicate. (G-I) KPC2 cells were treated with olaparib (5µmol/L) one hour before various doses of radiation (2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, 2 GyX3). Cells were collected 72 hours after treatment and RNA was extracted. qRT-PCR was conducted for Ifnβ (G), Cxcl9 (H), and Cxcl10 (I). Data are the mean fold change ± SEM relative to control treatment and represent 3 independent experiments performed in technical triplicate. (J, K) qRT-PCR targeting Mx1 in KPC2 (J) and mT4 (K) cells. Data are shown as mean fold change ± SEM relative to the control treatment and represent 4 (J) or 3 (K) independent experiments performed in technical triplicate. (L, M) Protein abundance quantification of CXCL9 (L) and CXCL10 (M) via ELISA in the media from KPC2 cells 72 hours after treatment with olaparib (3µmol/L or 5µmol/L) and/or radiation. Herring testis DNA (HS DNA) was transfected into cells (2µg) as a positive control. Data are average protein abundance ± SD. The graphs presented are representative of 2 independent experiments run in technical triplicate. (N) KPC2 cells treated in the absence or presence of IFNAR1 blocking antibody (5 µg/mL). Cells were collected 72 hours later and analyzed via flow cytometry for surface PD-L1 expression. Data are shown as MFI ± SEM relative to control treatment and represent 3 independent experiments performed in technical triplicate. (O-S) Flow cytometry analysis of PD-L1 (O, P) or MHC-I (Q-S) surface expression on MiaPaCa2 (O, R), AsPC-1 (P, S), and Panc-1 (Q). Data are shown as MFI ± SEM relative to control treatment and represent 1 (P, S), 2 (O, R) or 3 (Q) independent experiments performed in technical triplicate. Statistical significance was determined by a one-way ANOVA test (N) or a two-tailed, unpaired t test (A, G-I, J). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001