Supplementary Table 1

Transcription

1 Supplementary Table 1 Flow Cytometry Antibodies Antibody Fluorochrome Clone Vendor CD45 PE-cyanine 7 30-F11 D ioscience CD3 Pacific lue 17A2 iolegend (San Diego, CA) CD11b APC M1/70 iolegend (San Diego, CA) F4/80 FITC M8 eioscience, Inc (San Diego, CA) Ly6G PercP-cyanine 5.5 1A8 D ioscience Ly6C APC-cyanine 7 AL-21 D ioscience CD16/32 PE 93 eioscience, Inc (San Diego, CA) CD40 PE 3/23 D ioscience CD64 PE X54-5/7.1.1 D ioscience CD80 PE 16-10A1 D ioscience CD86 PE GL1 D ioscience I-A/I-E PE M5/ D ioscience IL-4Rα PE mil4r-m1 D ioscience OX40L PE RM134L D ioscience PD-L1 PE MIH5 D ioscience APC, allophycocyanin; PE, phycoerythrin, PercP, peridinin-chlorophyll protein complex; FITC, fluorescein isothiocyanate

2 Supplementary Figure 1 A. aseline Day +14 Diameter: 4.5 mm Volume: 95.0 mm 3 Diameter: 2.5 mm Volume: 54.2 mm 3. aseline Day +14 Diameter: 4.1 mm Volume: 47.0 mm 3 Diameter: 5.7 mm Volume: mm 3 Supplementary Figure 1. Ultrasound images showing representative examples of spontaneous pancreatic tumors from PC mice that (A) regressed or () progressed during therapy. PC mice were imaged and treated as described in Figure 5. oth examples are from mice treated with Gem+FG45+CEL. In the displayed images, the ultrasound detected tumor is highlighted in the right panel using a dotted line. Longest dimension diameter and volume for tumors is displayed below each set of images. = bowel, = kidney.

3 Supplementary Figure 2 H&E CD3 CD4 CD8 Gemcitabine + FG45 FG45 Gemcitabine Control Supplementary Figure 2. Treatment with gemcitabine and FG45 induces T cell infiltration into tumor tissue in a transplantation model of pancreatic carcinoma. Images showing H&E staining of tumors and immunohistochemistry for CD3, CD4, and CD8 expressing cells in tumors from mice described in Figure 1. Scale bars, 50 µm.

4 Supplementary Figure 3 H&E CD3 CD4 CD8 Foxp3 Gemcitabine + FG45 Non-responder Responder Control Supplementary Figure 3. T cells responding to treatment with gemcitabine and FG45 infiltrate the stromal tumor microenvironment. Images showing H&E staining and immunohistochemistry for CD3, CD4, CD8, and Foxp3 expressing cells in PDA explants re-implanted subcutaneously into PC mice and subsequently treated with IgG2a + PS (Control) versus gemcitabine + FG45 as described in Fig 3. Responder and non-responder indicate tumors which did and did not undergo regression, respectively, after treatment with gemcitabine plus FG45. Scale bars, 100 µm.

5 Supplementary Figure 4 H&E CD3 CD8 CD4 Foxp3 Gemcitabine + FG45 Control Supplementary Figure 4. Images showing H&E staining and CD3, CD4, CD8, and Foxp3 staining of spontaneously arising tumors from PC mice that have also been implanted subcutaneously with explanted tumor tissue and treated with or without gemcitabine and FG45 as described in Fig 3. Scale bars, 50 µm.

6 Supplementary Figure 5 A 100 CD3 100 Gem+FG+lysate Cells per 40x field Cells per 40x field CD4 CD8 Foxp3 C D E F CD3 CD4 CD8 Foxp3 Supplementary Figure 5. Delivery of tumor lysate in combination with gemcitabine and FG45 induces T cell infiltration into primary pancreatic tumors. (A) Whisker plots showing quantification by immunohistochemistry of CD3 cell infiltrates into pancreatic tumors of PC mice 14 days after the indicated treatment is shown (n = 4 per group). () Quantification of CD4, CD8, and Foxp3 expressing cells in pancreatic tumors from PC mice treated with gemcitabine + FG45 + tumor lysate administered subcutaneously (s.c.). Representative images showing immunohistochemistry for (C) CD3, (D) CD4, (E) CD8, and (F) Foxp3 expressing cells in pancreatic tumors from PC mice treated with gemcitabine + FG45 + tumor lysate.

7 Supplementary Figure 6 CD4 CD8 Foxp3 Ctrl CEL Gem + FG45 + CEL Supplementary Figure 6. Macrophages regulate T cell infiltration into spontaneously arising pancreatic tumors in the PC model. Representative images showing immunohistochemistry for CD4, CD8, and Foxp3 expressing cells in pancreatic tumors from PC mice 14 days after treatment with IgG2a + PS (Ctrl), clodronate encapsulated liposomes (CEL), and gemcitabine + FG45 + CEL. Scale bars, 50 µm.

8 Supplementary Figure 7 Control FG45 FG45 + CEL A C D FAP EpCAM DAPI FAP EpCAM DAPI FAP EpCAM DAPI Number of FAP + cells per 20x field Control FG45 FG45 + CEL Supplemental Figure 7. Treatment with an agonist CD40 mab with or without macrophage depletion does not impact the presence of cancer associated fibroblasts in PDAC. Images showing immunofluorescence staining of FAP + cancer associated fibroblasts (red), EpCAM + tumor cells (green), and DAPI stained nuclei (blue) in spontaneously arising tumors from PC mice receiving (A) control, () FG45, or (C) FG45 + CEL. (D) The number of FAP + stromal cells per 20x field is shown for each treatment group. P > 0.05 for comparisons between groups, Student s t test. Treatment Group

9 Supplementary Figure SSC-A FSC-A 95 SSC-A 100 SSC-A FSC-A FSC-H SSC-H live/dead CD11b CD11b 22 CD11b SSC-A F4/80 Ly6G CD3 CD F4/80 Ly6C Supplementary Figure 8. Gating strategy for identification of macrophages by flow cytometry. Shown are representative images from the analysis of splenocytes from a PC mouse with a primary pancreatic tumor. Mature macrophages are defined as CD45 + CD11b + CD3 neg Ly6G neg F4/80 + Ly6C low.