Synergy of radiotherapy and PD-1 blockade in Kras-mutant lung cancer

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1 Supplementary Information Synergy of radiotherapy and PD-1 blockade in Kras-mutant lung cancer Grit S. Herter-Sprie, Shohei Koyama, Houari Korideck, Josephine Hai, Jiehui Deng, Yvonne Y. Li, Kevin A. Buczkowski, Aaron K. Grant, Soumya Ullas, Kevin Rhee, Jillian D. Cavanaugh, Neermala Poudel Neupane, Camilla L. Christensen, Jan M. Herter, G. Mike Makrigiorgos, F. Stephen Hodi, Gordon J. Freeman, Glenn Dranoff, Peter S. Hammerman, Alec C. Kimmelman, and Kwok-Kin Wong Supplementary Figure 1 5 Supplementary ables 1

2 Supplementary Figure 1 A H Pre-treatment 8 weeks post 14 weeks post B Pre-treatment H 8 weeks post 2 weeks post L Supplementary Figure 1 umor growth kinetics in response to R and αpd-1 treatment in Kras-mutant murine NSCLC. (A) Representative MR images of a responsive (left lung) Kras-driven tumor at different time points (baseline, 8, and 14 weeks post treatment initiation). Contralateral tumor growth in the right lung (n=7). (B) Representative MR images of a resistant (left lung) Kras-driven tumor at different time points (baseline, 8, and 2 weeks post treatment initiation) (n=3). H (heart) circled in red. L (liver) circled in green. (tumor) circled with white/blue dotted line. 2

3 Supplementary Figure 2 A R refractory + αpd-1 R refractory B R refractory + αpd-1 R refractory cell exhaustion/inhibition cell recruitment/ activation Btla Eomes Ctla4 Lag3 Pd-1 Il1b Ccl5 Ifng Gzmb log2 M1-associated genes M2-associated genes Lrp1 Irf4 Mrc1 Socs1 Cd36 Arg1 gfb1 Il13 Il1 Il4 Ifnb1 Ifna4 Ifna2 Ifna1 Vegfc Vegfa Il6 nf Il23a Il12b Il12a Nos log2 Supplementary Figure 2 reatment of R-refractory K tumors with αpd-1 induces marker of cell inhibition. (A) Expression of selected cell-associated genes in Rrelapsed and R-relapsed αpd-1-treated tumors. (B) Expression of selected macrophage-associated genes in R-relapsed and R-relapsed αpd-1-treated tumors. Representative data are shown from mouse ncounter PanCancer Immune Profiling Panel conducted with RNA from 4 R-treated and 4 R-refractory αpd-1-treated mice (A, and B). Data are represented as mean ± SEM. Btla, B- and -lymphocyte attenuator; Eomes, Eomesodermin; Il1b, Interleukin 1 beta; Ccl5, Chemokine (C-C motif) ligand 5; Ifng, Interferon gamma; Gzmb, granzyme B; Lrp1, Low density lipoprotein receptor-related protein 1; Irf4, Interferon regulatory factor 4; Mrc1, Mannose receptor, C ype 1; Socs1, Suppressor of cytokine signaling 1; CD36, CD36 Molecule/hrombospondin receptor; Arg1, Arginase 1; gfb1, transforming growth factor beta-1; Il13, Interleukin 13; Il1, Interleukin 1; Il4, Interleukin 4; Ifnb1, Interferon beta 1; Ifna4, Interferon alpha 4; Ifna2, Interferon alpha 2; Ifna1, Interferon alpha 1; Vegfc, Vascular endothelial growth factor C; Vegfa, Vascular endothelial growth factor A; Il6, Interleukin 6; nf, umor necrosis factor; Il23a, Interleukin 23 alpha; Il12b, Interleukin 12 beta; Il12a, Interleukin 12 alpha; Nos2, Nitric oxide synthase 2. 3

4 Supplementary Figure 3 reatment response to R of Kras tumors 3 umour volume change (%) Weeks Supplementary Figure 3 umor growth kinetics in response to R in Kras-mutant murine NSCLC. umor volume kinetics of R-treated tumors. Each line represents one mouse (n=5). Data of this cohort was previously published (Herter-Sprie et al., 214). 4

5 Supplementary Figure 4 A CD45+ /EpCAM+ ratio [per mg tumor] unirradiated αpd-1 R+αPD-1 R B H Pre-treatment 4 weeks post R + αpd-1 6 weeks post R + αpd-1 D count/mg 3 Kras Kras/Lkb1 ** * 2 1 AM AN 8 % on CD8+ cells 4 Markers on tumor infiltrating CD8+ cells 6 * ** ** 4 2 Pd-1 Lag3 im3 6 % on CD4+ cells C Ctla4 Markers on tumor infiltrating CD4+ cells 4 2 Pd-1 Lag3 im3 Ctla4 Supplementary Figure 4 umor growth kinetics in response to R and αpd-1 treatment in Kras/Lkb1-mutant murine NSCLC and differences in tumor-associated immune cell populations compared to Kras-mutant murine NSCLC. (A) CD45+/EpCAM+ ratio calculated from Figure 5B (3 unirradiated, 3 R-treated, 3 αpd-1-treated, and 4 R+αPD-1-treated mice). (B) MR images of a progressive Kras/Lkb1 tumor at different time points (baseline, 4, and 6 weeks post treatment initiation) (n=2). H (heart) circled in red. (tumor) circled with white/blue dotted line. (C) Representative flow cytometry data (live/single/total CD45+ cells). otal numbers of tumor-infiltrating myeloid cells of unirradiated Kras and Kras/Lkb1 tumors (taken from Figure 2B and Figure 5B). (D) Expression of inhibitory cell markers on CD8+ and CD4+ cells of unirradiated Kras and Kras/Lkb1 tumors (taken from Figure 2E and Figure 5D). Representative data are shown conducted with 5 unirradiated Kras, and 3 unirradiated Kras/Lkb1 mice (C and D). Data are represented as mean ± SEM. P values were calculated using two-tailed Student s t-test. ***P<.1, **P<.1, *P<.5. 5

6 Supplementary Figure 5 adapted from Koyama et al., Cancer Res., 216 Alveolar macrophages (umor-associated macrophages: AM) umor cells CD13 + DC CD45 CD11c Live/Single cell gating Neutrophils (umor-associated neutrophils: AN) CD13 EpCAM or SSC-A CD45 CD11c Ly-6G CD19 FSC-A FSC-A CD11b CD11b CXCR2 NKp46 CD3 SSC-A SSC-A CD8a DX5 FSC-A Ly-6C Siglec-F CCR3 CD4 CD3 Eosinophils Ly-6C hi monocytes CD8 + cells NK cells Other myeloid cells (CD11b + DC, tissue macrophages, Ly-6C lo monocytes, etc.) 6 B cells CD4 + cells

7 Supplementary able 1 Antigen Clone Source Antigen Clone Source CD45 3-F11 BioLegend Siglec F E5-244 BD Biosciences CD3ε 145-2C11 BioLegend PD-1 29F.1A12 BioLegend CD4 RM4-5 BioLegend IM-3 RM3-23 BioLegend CD8a BioLegend LAG-3 C9B7W BioLegend CD19 6D5 BioLegend CD13 2E7 BioLegend DX5 DX5 BioLegend PD-L1 1F.9G2 BioLegend NKp46 29A1.4 BioLegend EpCAM G8.8 BioLegend CD11c N418 BioLegend CD16/32 2.4G2 BioLegend CD11b M1/7 BioLegend Ly-6G 1A8 BioLegend FOXP3 FJK-16s ebioscience Ly-6C HK1.4 BioLegend CLA-4 UC1-4B9 ebioscience Supplementary able 1 List of murine antibodies used for flow cytometry analysis. 7