Supplementary Figure 1 A green: cytokeratin 8

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1 Supplementary Figure 1 A green: cytokeratin 8 green: α-sma red: α-sma blue: DAPI blue: DAPI Panc-1 Panc-1 Panc-1+hPSC Panc-1+hPSC monoculture coculture B Suppl. Figure 1: A, Immunofluorescence staining of clonogenic survival assays using the PCC and PSC specific markers cytokeratin 8 and α-sma, respectively. PSC are present around the PCC colonies and not within them. B, Cell cycle distribution determined by flow cytometry of PCC in monoculture or coculture with hpsc. hpsc is a human pancreatic stellate cell line isolated and established from the resected pancreas of a patient undergoing operation for pancreatic cancer. This cell line expresses several markers of activated PSCs including alpha-sma, fibronectin, vimentin, and type I collagen, and considered as spontaneously immortalized after having grown in culture more than a year. The detailed characteristics of the hpsc will be described in a separate manuscript. Cell culture conditions: hpsc was cultured in Ham s F-12/DMEM medium and the remaining cell lines in DMEM. Culture medium was supplemented with 10% heat-inactivated Foetal Calf Serum (FCS), penicillin sodium and streptomycin sulfate. Cells were maintained in a humidified atmosphere containing 5% CO 2 at 37 C.

2 Supplementary Figure 2 A B 1 hpsc LTC-14 * 0.1 C Mia 0.93 Mia+hPSC 0.86 R 1.18 D hpsc 0.05 LTC Dose (Gy) E F * * Suppl. Figure 2: A, Clonogenic survival curve of MiaPaCa-2 in coculture with hpsc. *, F- test P= : plating efficiency, R: protection enhancement ratio. B, Clonogenic survival of PSC (hpsc and LTC-14) in monoculture. C-D, Ham s F-12/DMEM medium + 10% FBS was conditioned on confluent hpsc for 48 hours. The medium was hereafter filtrated and used 1:1 with fresh medium to seed PCC for clonogenic survival assays. Two hours post irradiation the conditioned medium was diluted down to 1:3 with fresh medium. C, Panc-1 and D, PSN-1. E-F, For feeder layer experiments hpsc were seeded and three hours later exposed to 10 Gy, a dose with a predicted survival of %. PCC were hereafter seeded on top and irradiated accordingly. E, Panc-1 and F, PSN-1. *: F test, P<0.05. All graphs are representative of two independent experiments. Error bars denote SE.

3 Supplementary Figure 3 A B C Suppl. Figure 3: DNA damage induced 53BP1 foci in mono- and coculture. Cocultured PCC and PSC were irradiated, fixed after 1h, 6h and 24h, and immunostained for 53BP1 here in red (Cell Signaling), Cytokeratin 8 (Thermo Scientific) and DAPI (A). Foci were detected using an IN Cell Analyzer 1000 automated epifluorescence microscope (GE Healthcare) and quantitation performed using IN Cell Analyzer Workstation software. Only Cytokeratin 8 (epithelial cell marker) positive cells were included in analysis. B, Panc-1 monocultured or cocultured with hpsc or LTC-14 C, PSN-1asinB. Error bars denote standard deviation. Graph are representative of three independent experiments.

4 Supplementary Figure 4 A B C Panc P+hPSC 0.46 P+β1 ab 0.52 P+hPSC+β1 ab 0.52 Panc P+hPSC 0.47 P+β P+hPSC+β R Panc P+hPSC 0.63 P+LY 0.52 P+hPSC+LY 0.61 R D E Panc P+hPSC 0.55 P+FAK 0.23 P+hPSC+FAK 0.35 R Panc P+hPSC 0.47 P+β1/FAK 0.25 P+hPSC+β1/FAK 0.34 R Suppl. Figure 4: Plating efficiency and protection enhancement ratio. The experimental data were fitted with the linear quadratic model: S = exp(-αd βd 2 ) Where S is survival probability, D is the radiation dose (Gy) and α and β are the fit parameters (Gy -1 and Gy -2, respectively). The protection enhancement ratio (R) was used to quantify radioprotection (R 10 was calculated as R 10 = D treated /D control, where D treated and D control doses yield 10% survival for treated and control cells, respectively). One hundred cells were seeded at 0 Gy and this number doubled for each 2 Gy increment in radiation dose. The plating efficiency () and R are given for clonogenic survival assays presented in A, Figure 3A. Since just two dose points was investigated in this experiment it is not possible to calculate the R 10. B, Figure 3C, C, Figure 4A, D, Figure 4B, E, Figure 4C.

5 Supplementary Figure 5 A B C D Suppl. Figure 5: Surface expression of β1-integrin in PCC and PSC in mono- and coculture. PSC were labelled with Vybrant Cell-Labelling DiI for 20 min in serum-free medium at 37 C followed by 3x washes in medium+serum prior to coculture with PCC. Samples were trypsinized and stained with 10 µl anti-β1-integrin FITC antibody (MCA1949F, Serotec) for 30 min on ice in the dark. Samples were analyzed by flow cytometry. A, Panc-1 4 hr after plating B, Panc-1 24 hrs after plating C, hpsc 24 hrs after plating and D, PSN-1 24 hrs after plating. P values derived from t-test. RFU: Relative Fluorescence Units.

6 Supplementary Figure 6 A B C 250 µm 250 µm 250 µm D Suppl. Figure 6: PSC contribution to the tumor at time of randomization. Female nude mice were injected subcutaneously into the flank with with either 1*10 6 PSN-1 or 1*10 6 PSN-1 + 4*10 6 LTC-14; n=5 animals. LTC-14 are immortalized by means of SV40 large T antigen. Tumors were snap frozen once they reached 50mm 3 volume and sectioned into 10µm slices. Slices were fixed in ice cold methanol for 15 min and after blocking of endogenous peroxidase activity with 3% hydrogen peroxide for 20 min, the sections were sequential treated with avidin, biotin (Avidin Biotin blocking kit; Vector Laboratories Inc.) and Tris-NaCl-blocking buffer. Overnight incubation at 4 C with primary biotin mouse anti-sv40 large T and small t Antigens antibody (#554151, BD Pharmingen) was followed by avidin-biotin-peroxidase reaction (Vectastain ABC ELITE Kit; Vector Laboratories Inc.). The complex was visualised with 3,3 diaminobenzidene (ImmPACT DAB Peroxidase Substrate, Vector Laboratories Inc.) and tissues were counterstained with haematoxylin. A- B, Representative microphotograph of immunohistochemistry staining of SV40 large T antigen (SV40 T-Ag) on PSN-1+LTC-14 (A) and on PSN-1 (B) tumor biopsy. SV40 T-Ag staining highlights g LTC-14 cells. C, Negative control performed by omitting primary antibody. D, The graph shows the percentage of SV40 T-Ag positive cells within each tumour. The percentage was evaluated by manually counting five fields per slides and it ranges between 4.7% and 9.4% (average 7.1% ) of SV40 T-Ag positive cells per tumor.

7 Supplementary Figure 7 Suppl. Figure 7: Tumor growth curves. Mice were injected with either 1*10 6 PSN-1 (1M) or 5*10 6 PSN-1 (5M). When tumor volume reached 50 mm 3 the animals were randomised and radiated with a single dose of 6 Gy (XRT). Tumor volume was measured by callipers every 2-3 days hereafter. n=4-6 animals per group.