Supplemental Materials. Stromal Modulation Reverses Primary Resistance to Immune Checkpoint Blockade in. Pancreatic Cancer.
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1 Supplemental Materials Stromal Modulation Reverses Primary Resistance to Immune Checkpoint Blockade in Pancreatic Cancer Jun Zhao 1, Zhilan Xiao 2, 3, Tingting Li 1, 4, Huiqin Chen 5, Ying Yuan 5, Alan Y. Wang 6, Cheng-Hui Hsiao 7, Diana S-L Chow 7, Willem W. Overwijk 2, 8, * 1, 8, *, Chun Li 1 Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, Texas 77054, USA. 2 Department of Melanoma Medical Oncology - Research, The University of Texas MD Anderson Cancer Center, Houston, Texas 77054, USA. 3 West China School of Medicine/West China Hospital, Sichuan University, Chengdu , Sichuan, China 4 Department of Biophysics, School of Life Science & Technology, University of Electronic Science and Technology of China, Chengdu , Sichuan, China 5 Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77054, USA. 6 Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77054, USA. 7 Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77030, USA 8 The University of Texas MD Anderson Cancer Center, UTHealth Graduate School of Biomedical Sciences at Houston, Houston, TX 77054, USA Corresponding Authors: Chun Li, cli@mdanderson.org Willem W. Overwijk, woverwijk@mdanderson.org
2 Supplemental Figures Figure S1. Relative mrna expression of hedgehog, stemness, and hypoxia markers in M- CPA/PTX-treated Kras* tumors. Results are mean ± SEM of 3 mice in each group. RT-PCR was performed in technical duplicates, and values were normalized to 18S.
3 Figure S2. H&E sections from non-tumor organs. Tissues were collected from mice bearing Kras* tumor at 10 days after the treatment started. Scale bar = 50 µm.
4 Figure S3. Representative images of Kras* orthotopic tumors. (A) Untreated control tumor before resection (blue arrow). (B) Representative tumors from control, anti-pd1, M-CPA/PTX, and combination group. Tumors were collected at 10 days after enrollment, at which time, 3 doses of M-CPA/PTX and/or 4 doses of anti-pd1 were injected.
5 Figure S4. Representative flow charts and gating strategy for dendritic cells, MDSCs, and macrophages. Dendritic cells: single/live cell/cd45 + /CD11b + /CD11c +. MDSC: single/live cell/cd45 + /CD11b + /Ly6G + or single/live cell/cd45 + /CD11b + /Ly6C +. Macrophages: single/live cell/cd45 + /CD11b + /F4/80 +.
6 Figure S5. Representative flow charts and gating strategy for T cell populations. CD8 T cells: single/live cell/cd45 + /CD8 +. Tregs: single/live cell/cd45 + /CD4 + /Foxp3 +.
7 Figure S6. Relative mrna expression of IFN-γ, PD-L1, and CTLA-4 in Kras* orthotopic tumor. Results are mean ± SEM of 3 mice in each group. RT-PCR was performed in technical duplicates, and values were normalized to 18S.
8 Control M-CPA/PTX Anti-PD1 M-CPA/PTX + Anti-PD1 Figure S7. Analyses of intratumoral cytokines after treatment. Data are presented as mean ± SEM (N = 5). Significance was determined using 1-way ANOVA followed by Tukey post hoc analysis. *p < 0.05, ns = not significant.
9 Figure S8. Representative images of Kras* orthotopic tumors after anti-ifn-γ neutralization. Mice bearing Kras* orthotopic tumors were enrolled once tumors were palpable, and assigned to untreated control, M-CPA/PTX + anti-pd1, or M-CPA/PTX + anti-pd1 + anti-ifn-γ groups. M- CPA/PTX was intravenously injected at 5 mg/kg/drug on days 0, 1, and 2. Anti-PD1 was intraperitoneally injected at 100 µg per dose on days 4, 6, 8, and 10. Anti-IFN-γ was intraperitoneally injected at 200 µg per dose on days 4, 6, 8, and 10. Tumors were collected at 10 days after the initiation of treatment. Three mice were included in each group.
10 Figure S9. Representative images of combination-treated Kras* tumor sections costained with CD31 (red) and CD8α (green). CD8α + T cells extravasated from blood vessels and penetrated into tumor mass. Scale bar = 50 µm. Figure S10. Intratumoral drug concentration after 3 daily doses of M-CPA/PTX at 5 mg/kg/drug per injection. Tumors were collected at 24 hours after the last dose of M- CPA/PTX Results are mean ± SEM of 4 tumors in each group.
11 Figure S11. Kras* viability. (A) Cell viability after treatment with CPA or micelles-encapsulated CPA (M-CPA). (B) Cell viability after treatment with PTX and/or IFN-γ. Cells were treated at 37C for 72 hours. N= 6 each group. Data are presented as mean ± SEM.
12 Figure S12. IFN-γ receptor is required for the additive cell killing effect of combined IFN-γ + PTX. (A) Immunofluorescence imaging of IFN-γ receptor in Kras* cells and corresponding cell viability assay. (B) Immunofluorescence imaging of IFN-γ receptor in Panc-1 cells and corresponding cell viability assay. Cell viability was expressed as number of viable cells in treatment groups as a percentage of cells in untreated control group. Data are presented as mean ± SEM. N = 6 per group.
13 Figure S13. IFN-γ did not induce significant apoptosis in Kras* cell culture. Cells were treated at 37 C for 48 hours.
14 Figure S14. Reverse-phase protein array analyses of PTX and IFN-γ combination. Antibody targets with expression in PTX + IFN-γ group > 1.3 times or < 0.77 times expression in control group.
15 Figure S15. Western blot of Kras* cells after treatment with CPA, PTX, and/or IFN-γ. Kras* cells were treated with CPA (1 µm), PTX (10 nm), IFN-γ (10 ng/ml), or combinations of the two or three reagents for 48 hours at 37 C. Two runs of experiments were performed.
16 Figure S16. Representative bioluminescence images of KPC-Luc mice. Mice in the control, anti-pd1, and M-CPA/PTX groups were imaged at 2 weeks after enrollment. Mice in the M- CPA/PTX + anti-pd1 group were imaged at 4 weeks after enrollment, at which time both M- CPA/PTX and anti-pd1 had been injected. Four mice were included for each group.
17 Supplemental Table 1. Primary antibody information Target Host Source and Catalog Number Application CD31 Rabbit Abcam, IHC Ki67 (mouse specific) Rabbit Cell Signaling, #12202 IHC α-smooth muscle actin Rabbit Abcam, ab5694 IHC CD8α Rat BioLegend, IHC IFN-γ Rabbit Novus Biologicals, NBP IHC Shh Rabbit Santa Cruz, sc-9024 IHC, WB Carbonic anhydrases IX (N-19) Fibroblast activation protein alpha Goat Santa Cruz, sc WB Rabbit Novus Biologicals, NB WB Angiogenesis kit Rabbit Cell Signaling, 8696T WB Supplemental Table 2. RT-PCR Primers (Mouse) Target Sequence (5 to 3 ) Reference 18S PD-L1 IFN-γ CTLA-4 Forward: GTAACCCGTTGAACCCCATT Reverse: CCATCCAATCGGTAGTAGCG Forward: GCTCCAAAGGACTTGTACGTG Reverse: TGATCTGAAGGGCAGCATTTC Forward: ATGAACGCTACACACTGCATC Reverse: CCATCCTTTTGCCAGTTCCTC Forward: TTTTGTAGCCCTGCTCACTCT Reverse: CTGAAGGTTGGGTCACCTGTA Ozdemir et al, Cancer Cell 2014 Primer Bank a1 Primer Bank a1 Primer Bank a1
18 Gli1 Smo Forward: CCAAGCCAACTTTATGTCAGGG Reverse: AGCCCGCTTCTTTGTTAATTTGA Forward: GAGCGTAGCTTCCGGGACTA Reverse: CTGGGCCGATTCTTGATCTCA Reference 4 PrimerBank: a1 Ptch1 Forward: AAAGAACTGCGGCAAGTTTTTG Reverse: CTTCTCCTATCTTCTGACGGGT PrimerBank: a1 Shh Forward: AAAGCTGACCCCTTTAGCCTA Reverse: TTCGGAGTTTCTTGTGATCTTCC PrimerBank: a1 Nanog Aldh1a1 Forward: TCTTCCTGGTCCCCACAGTTT Reverse: GCAAGAATAGTTCTCGGGATGAA Forward: ATACTTGTCGGATTTAGGAGGCT Reverse: GGGCCTATCTTCCAAATGAACA PrimerBank: a1 PrimerBank: a1 Sox2 Forward: GCGGAGTGGAAACTTTTGTCC Reverse: CGGGAAGCGTGTACTTATCCTT PrimerBank: c1 Hif1a Forward: ACCTTCATCGGAAACTCCAAAG Reverse: CTGTTAGGCTGGGAAAAGTTAGG PrimerBank: a1 Caix Forward: TGCTCCAAGTGTCTGCTCAG Reverse: CAGGTGCATCCTCTTCACTGG PrimerBank: a1
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Supplementary Figure 1 a Percent of body weight! (%) 4! 3! 1! Epididymal fat Subcutaneous fat Liver SD Percent of body weight! (%) ** 3! 1! SD Percent of body weight! (%) 6! 4! SD ** b Blood glucose (mg/dl)!
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