Supplemental Information. Cancer-Associated Fibroblasts Neutralize. the Anti-tumor Effect of CSF1 Receptor Blockade
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1 Cancer Cell, Volume 32 Supplemental Information Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors Vinit Kumar, Laxminarasimha Donthireddy, Douglas Marvel, Thomas Condamine, Fang Wang, Sergio Lavilla-Alonso, Ayumi Hashimoto, Prashanthi Vonteddu, Reeti Behera, Marlee A. Goins, Charles Mulligan, Brian Nam, Neil Hockstein, Fred Denstman, Shanti Shakamuri, David W. Speicher, Ashani T. Weeraratna, Timothy Chao, Robert H. Vonderheide, Lucia R. Languino, Peter Ordentlich, Qin Liu, Xiaowei Xu, Albert Lo, Ellen Puré, Chunsheng Zhang, Andrey Loboda, Manuel A. Sepulveda, Linda A. Snyder, and Dmitry I. Gabrilovich
2 A CSF1R-inh Ly6C Ly6C F4/8 F4/8 Ly6G Ly6G Ly6G Ly6G B CSF1R-inh C D B16-F1 TAM PMN-MDSC M-MDSC EL-4 TAM PMN-MDSC M-MDSC LLC TAM PMN-MDSC M-MDSC CSF-1R-inh cells per gram tumor (x1 3 ) Cells/mm 2 E F 5 TAM Isotype Cntrl CD115 Ab PMN-MDSC % of live cells Isotype CD Days Post Tumor Injection Isotype CD115 G H Isotype CD115 % of live cells % of live cells % of live cells % of live leukoocytes Isotypel CD115 Isotypel CD115
3 Figure S1. Related to Figure 1: PMN-MDSC in tumor tissues of mice treated with CSF-1R inhibitor. A. A Typical example of staining of tumor tissues of TRAMP mice treated with vehicle (control) or JNJ (CSF-1R-inh). B. A Typical example of staining of tumor tissues with Ly6G antibody. Scale bar = 5 µm. C. The number of Ly6G+ cells per mm2 in mice treated with JNJ Each group included 4 mice. - p<.5 between groups. D. The number of myeloid cells in tumor tissues of mice treated with JNJ (CSF1R inh) or vehicle (control) 1 days after tumor inoculation. Tumor model is indicated on the graph. Each group included 4 mice. - p<.5; - p<.1 between groups. E. Effect of 3-week treatment of CD115 ab (25 µg/mouse, twice a week) on tumor growth in LLC tumor model (n=5). F. Proportion of myeloid cells in tumors. G. Proportion of myeloid cells in BM and spleens of the mice. Mean and SD from 4 mice are shown. - p<.5; - p<.1 between groups. H. Proportion of M-MDSC in different tissues of mice treated with CSF-1R inhibitor (n=4). In all experiments Mean and SD are shown. p values were calculated using Student s t-test.
4 F4/8 - Gr1 + CD11b + Gr1 + CD11b + Ly6C lo Ly6G + F4/8 + Gr1 - A B Percent of Live Cells C 8 6 No Treatment CSFR1-inh D 4 2 F4/8 + Gr1 - F4/8 - Gr1 + CD11b + Gr No Treatment CSFR1-inh # of Cells Percent of Live Cells # of Cells CD11b + Ly6C lo Ly6G + CD11b + Gr1 + F4/8 - Gr1 + F4/8 + Gr1 - E 25 2 No Treatment CSFR1-inh F4/8 + Gr1 - F4/8 - Gr1 + CD11b + Gr1 + CD11b + Ly6C lo Ly6G + F4/8 + Gr1 - F4/8 - Gr1 + CD11b + Gr1 + F CD11b + Ly6C lo Ly6G +
5 Figure S2. Related to Figure 2: Effect of JNJ on myeloid cell differentiation. A-D. Percentage and absolute number of cells generated from enriched BM HPC during 3-day (A,B) or6-day(c,d) culture with GM-CSF and JNJ E, F Percentage and absolute number of cells generated from enriched BM HPC without treatment with TES. 3 days (E) and 6 days (F). Each group included 3 replicates. - p<.5; - p<.1 from no JNJ group.
6 A MEP GMP Day 9 Day 16 Day 23 Day 9 Day 16 Day 23 B 4 3 Day 9 Vehicle ctrl CSFR1-inh 2 1 CFU-GM CFU-M C 8 LLC Geo mean CSF1R-inh
7 Figure S3. Related to Figure 2: Effect of JNJ on myeloid progenitors. A. Effect of JNJ on CMP and GMP in BM at different time during the treatment. B. Effect of JNJ treatment on BM cell colony formation (n=3). Treatment was performed as described in Figure 2. C. Expression of CXCR2 on BM PMN-MDSC of mice treated with CSF1R inhibitor for 3 weeks. Each experiment included 3 mice. - p<.5; between groups.
8 A D Tumor cells CAF B B16F1 LLC 4T1 EL-4 CSF 1 isotype C pg/2 ul of Supernatant G E H F KO WT pg/1 ug of protein
9 Figure S4. Related to Figure 4. Deletion of CSF1 in tumor cells. A. Typical gating and sorting strategy for isolation of fibroblasts. B. Expression of CXCR2 on neutrophils cultured for 24 hr with CSF1. C. Expression of CXCR2 on neutrophils in peripheral blood of mice 5 weeks after reconstitution with wild-type (WT) and CXCR2-KO bone marrow. D. Expression of csf1 mrna (qpcr) in CAF and tumor cells in LLC-TB mice (n=3). E. Expression of csf1 mrna (qpcr) in different tumor cell lines. F. csf1 expression in different clones of LLC tumor cells after CRISPR-CAS9 deletion of csf1. G. Clone of LLC tumor cells lacking Csf1 production after 3 weeks of tumor growth in vivo. H. Tumor growth of WT and M-CSF deleted LLC tumor cells (n=5).
10 A B K Relative expression Relative expression Relative expression C D E F G H I J Relative expression
11 Figure S5. Related to Figure 5: CSF1 shrna in fibroblasts. A, B. Mouse lung fibroblasts transduced with different csf1r constructs. Expression of csf1r (A) and pdgfra (B) are shown. Constructs and were used in the studies. - lentivirus with empty vector. C, D. CAF isolated from tumors of LLC TB mice were cultured with Csf1 (2 ng/ml) in the presence or absence of Csf1R inh (C) or Csf1 neutralizing Ab (5ug/ml) (D) for 4 days. Cxcl1 protein in supernatants was measured by ELISA. Cumulative results of three experiments (Mean and SD) are shown. E,F. CAF isolated from tumors of LLC TB mice were treated with CSF-1 alone or with either CSF-1R inh (E) or CSF-1 neutralizing antibody (F). Supernatants collected from culture were used for BM neutrophils chemotaxis. Mean and SD, n=3 are shown. p<.5; p<.1. G. CAF were transduced either with control vector or Csf1r shrna construct. Expression of csf-1r was analyzed by qpcr. β-actin was used as a loading control. H. CAF transduced either with lentiviral control vector or Csf1R shrna construct were treated with Csf1 for 4 days. Supernatants obtained from culture were used for BM PMN-MDSC chemotaxis. Mean and SD, n=3 are shown. p<.1. I. Gatingstrategyfor analysis of cells in human tumors; J, K. Human fibroblasts from NSCLC patient transduced with different CSF1R shrna constructs. Expression of CSF1R (J) and PDGFAR (K) are shown. Constructs 1644 and were used in the studies. Construct was used as additional control. - lentivirus with empty vector. - significant (p<.5) differences from control.
12 A B 1 p= Above median Below median blood PMN-MDSC (%) Above median p=.21 Below Median blood M-MDSC (%) Above median p=.9 Below median C Low score staining High score staining
13 Figure S6. Related to Figure 6: MDSC in peripheral blood of cancer patients. A. Proportion of PMN-MDSC (left) and M-MDSC (right) in peripheral blood of samples split based on CXCL8 levels in tumors. B. Proportion of PMN-MDSC (left) and M-MDSC (right) in peripheral blood of samples split based on CSF-1 levels in tumors. C. Examples of staining of tissues for CSF-1. Scale bar 5 µm.
14 Table S1, related to STAR Methods Sequence of primers used for qpcr Murine Mif F CAT CGC AGT GCA CGT GGT CC; R GGC ACC ACC GAT CTT GCC GA cxcl 1 F CGA GGC TTG CCT TGA CCC TGA A; R GGG TCA TAT GCC AGT CCC ACC cxcl 2 F TTT GCC TTG ACC CTG AAG CCC C; R TTC CGT TGA GGG ACA GCA GCC cxcl 5 F GCA TCC CCA GCG GTT CCA TCT; R GCT CCG TTG CGG CTA TGA CTG A cxcl 7 F CTC AGA CCT ACA TCG TCC TGC ACC; R CTG TAA GGG GAG CCA GCG CA cxcl 12 F GAG GAA GGC TGA CAT CCG TGG G; R ATG ACC CCA GTC AGT GCT GTC C Csf 1 F CATCCAGGCAGAGACTGACA; R CTTGCTGATCCTCCTTCCAG ccl 2 F CCCAATGAGTAGGCTGGAGA; R AAAATGGATCCACACCTTGC ccl 3 F CCA AGT CTT CTC AGC GCC ATA 3' ; R GAT GAA TTG GCG TGG AAT CTT C 3' ccl 4 F TGC TCG TGG CTG CCT TCT 3' ; R CTG CCG GGA GGT GTA AGA GA 3' ccl 5 F TGC CCA CGT CAA GGA GTA TT 3 ; R CAG GAC CGG AGT GGG AGT A 3 ccl 6 F 5 ATGAGAAACTCCAAGACTGCC 3 ; R 5 TTATTGGAGGGTTATAGCGACG 3 ccl 7 F CCA AGT GTG GGC CCA ACC AGA T; R GCT TCC CAG GGA CAC CGA CT pdgfra F AGAAAATCCGATACCCGGAG; R AGAGGAGGAGCTTGAGGGAG csf 1r F TTGCCTTCGTATCTCTCGATG; R CTCTGCTGGTGCTACTGCTG β actin F ACCGCTCGTTGCCAATAGTGATGA 3 ; R TGAGAGGGAAATCGTGCGTGACAT 3 Human Cxcl 8 F GACCACACTGCGCCAACAC; R CTTCTCCACAACCCTCTGCAC Pdgfra F GCTCAGCCCTGTGAGAAGAC; R ATTGCGGAATAACATCGGAG Csf 1r F TTGGGCTGATCCTCTTCTTC; R AAAGCGTGAGAGCACGAAGT Gapdh F GCATCTTCTTTTGCGTCG; R GACCAAATCCGTTGACTC
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Supplementary Figure 1 a
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Supplementary Materials 1 Supplementary Table 1. List of primers used for quantitative PCR analysis. Gene name Gene symbol Accession IDs Sequence range Product Primer sequences size (bp) β-actin Actb gi
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