iNOS regulates the therapeutic response of pancreatic cancer cells to radiation

therapy

Patricia M. R. Pereira1, Kimberly J. Edwards1, Komal Mandleywala1, Lukas M. Carter1,

Freddy E. Escorcia2, Luis Felipe Campesato3, Mike Cornejo1, Lolkje Abma1, Abu-Akeel

Mohsen3, Christine A. Iacobuzio-Donahue4,5, Taha Merghoub3,6, Jason S. Lewis1,7,8,9,10*

Affiliations:

1 Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY

10065, USA

2 Molecular Imaging Program, Center for Cancer Research, National Cancer Institute,

National Institutes of Health, Bethesda, MD 20814, USA

3 Swim Across America and Ludwig Collaborative Laboratory, Immunology Program,

Parker Institute for Cancer Immunotherapy, Memorial Sloan Kettering Cancer Center,

New York, NY, United States.

4 The David M. Rubenstein Center for Pancreatic Cancer Research, Human Oncology and

Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

5 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, USA.

6 Department of Medicine, Weill Cornell Medical College, New York, NY, United States.

7 Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New

York, NY, USA

8 Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA

9 Department of Radiology, Weill Cornell Medical College, New York, NY, USA

10 Radiochemistry and Molecular Imaging Probes Core, Memorial Sloan Kettering Cancer

Center,New York, NY, USA

Corresponding author:

J.S.Lewis lewisj2@mskcc.org P: +1 646-888-3038, Fax: 646-888-3059

Supplementary Methods

Isolation and expansion of murine and human pancreatic fibroblasts

Briefly, at 2 days post-RT 12 Gy (see previous section for details on the RT protocol), the

mice were sacrificed and the pancreata were removed and incubated in ice-cold PBS

containing 2 μM trypsin inhibitor (Sigma-Aldrich). After cutting the tumors into small

pieces (≈ 2 mm3) using a scalpel, 15 of these pieces were attached to Petri dishes using 2

μL of murine blood serum mixed with 2 μL of 10 mM calcium chloride (CaCl2). To

induce fibroblasts migration from the tumor tissue (Supplementary Fig. 4), tissues were

cultured in DME medium containing 1 g L-1 glucose, 10 mM HEPES, 100 μg mL-1

gentamicin, 1% L-glutamine, 100 μg ml-1 Penicillin/Streptomycin, and 20% FCS. The

medium was replaced every two days. After 15 days in culture, the tissues were removed

and fibroblasts were detached using trypsin. Fibroblasts were then expanded in cell

culture flasks (Supplementary Fig. 4) using DME medium containing 1 g L-1 glucose,

10 mM HEPES, 100 μg mL-1 gentamicin, 1% L-glutamine, 100 μg ml-1

Penicillin/Streptomycin, and 10% FCS. Murine fibroblasts were used in this work within

passage number of three. The myofibroblastic phenotype of the cells used in this work

was confirmed before experiments using immunofluorescence staining for vimentin and

α-smooth muscle actin (Supplementary Fig. 4).

To isolate fibroblasts from a human PDAC, deidentified tumor tissues were obtained

from David M. Rubenstein Center for Pancreatic Cancer Research following IRB

approval. Human fibroblasts were isolated using the protocol previously described for

murine fibroblasts with some modifications: tumor pieces were attached to Petri dishes

using 2 μL of human blood serum mixed with 2 μL of 10 mM CaCl2. Fibroblasts

expansion was observed after 25 days in culture. Human fibroblasts were used in this

work within passage number of two.

In vitro therapeutic experiments

Radiation treatment (RT). Fibroblasts, FC1245 pancreatic cancer cells or co-cultures

were irradiated with a 137Cs irradiator (Shepherd Mark-I, model 68, SN 643) at a dose rate

of 5 Gy per minute. The 5 Gy RT protocol used in this work was based on previous

studies demonstrating that irradiated fibroblasts support the invasiveness of irradiated

pancreatic cancer cells [1] and that iNOS inhibition combined with RT is effective

against squamous carcinoma xenografts [2].

iNOS inhibition with 1400W. For iNOS inhibitory studies, vehicle or 10 μM N-(3-

(aminomethyl)benzyl)acetamidine (1400W; Cayman Chemicals) was added (a) 12 h

before RT, or (b) at 12 h before RT and immediately after RT.

iNOS knockdown and iNOS amplification. iNOS was depleted using a pool of three

target-specific 20–25 nt siRNA (Santa Cruz Biotechnology). FC1245 pancreatic cancer

cells were transfected in 6-well culture plates, at 60–80% confluence, with siRNA iNOS.

Cells were also transfected with a scrambled (scr) siRNA in parallel as controls. For each

transfection, cells were treated for 5 h with 2.4 μM of siRNA in transfection medium

(Santa Cruz) containing 0.5 μL cm−2 of transfection reagent (Santa Cruz). RT was

performed 24 h post-transfection with corresponding siRNA.

iNOS was amplified in FC1245 cells using NOS2 CRISPR activation plasmid (m, Santa

Cruz). Cancer cells were transfected in 6-well culture plates, at 60–80% confluence, with

CRISPR Activation Plasmid NOS2. For each transfection, cells were treated for 5 h with

0.9 ng μL−1 of CRISPR Activation Plasmid NOS2 in transfection medium (Santa Cruz)

containing 0.5 μL cm−2 of transfection reagent (Santa Cruz).

NO donor treatments. For NO donor studies, cells were treated with 1 μM DETA-

NONOate (Cayman Chemicals) for 6 h before experiments. The concentration of DETA-

NONOate used in our experiments was based on previous studies demonstrating that

concentrations of DETA-NONOate in the range of 20-2000 nM promote ovarian cancer

growth [3].

Glucose uptake. 24 h following RT, cells were incubated with 0.025 mM 2-(N-(7-

Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG; Abcam) in KREBS

buffer for 30 min. After washing with PBS, cells were lysed in 1% sodium dodecyl

sulfate (SDS; Sigma-Aldrich) solution and fluorescence was measured with excitation

and emission wavelengths at 488 and 650 nm, respectively. NBDG fluorescence levels

were normalized to cell numbers.

Lactate production. Lactate production was measured at 24 h after RT in the supernatant

of cells using a fluorescent L-lactate assay kit (Cayman Chemicals) according to

manufacturer instructions. Lactate values were normalized to cell numbers.

Cell counting and viability assessment with trypan blue exclusion. Cells were treated with

1400W, siRNA iNOS, DETA-NONOate, or scr siRNA for 24 h before RT experiments.

Twenty-four hours following irradiation, cells were harvested, dissociated by

trypsinization, mixed with trypan blue (1:1 volumes), and counted on an automated cell

counter.

Migration and invasion assays

Boyden chamber assay. Pancreatic cancer cell migration was evaluated in the presence

and absence of conditioned media obtained from irradiated and non-irradiated fibroblasts

using the Boyden Chamber assay. FC1245 PDAC cells in the presence of conditioned

media from fibroblasts were cultured in a 24-well format culture plate using a 3.0 μm

membrane filter (Corning). Following a 24 h incubation, cells were removed by wiping

from the top of the membrane, and the cells that migrated to the bottom were stained and

quantified by cell counting.

Scratch wound-healing assay. FC1245 PDAC cells were cultured in the presence of

conditioned media from fibroblasts in a 24-well cell culture plate (1.9 x 105 cells per

well) for 24 h. The cell monolayers were then gently and slowly scratched with a 1 mL

pipette tip across the center of the well. After scratching, monolayers were gently washed

with medium to remove detached cells. Cells were photographed at 0, 4, 21, 28, and 48 h

after scratch generation.

Invasion assay. FC1245 PDAC cells invasion in the presence and absence of conditioned

media from irradiated and non-irradiated fibroblasts was determined using matrigel

invasion chambers (Corning) according to manufacturer instructions. Following 24 h

incubation, the non-invading cells were removed from the upper surface of the membrane

by “scrubbing” with cotton tipped swab. The cells that migrated were fixed in 4%

paraformaldehyde (PFA, Santa Cruz Biotechnology), the membrane was removed from

the insert housing and mounted on a microscope slide using vectashield mounting

medium with DAPI (Vector Laboratories). Slides were scanned with Pannoramic MIDI

scanner (3DHistech, Hungary) using 20x/0.8NA objective lens. Regions of interest

around the cells were then drawn and exported as .tif files from these scans using

Caseviewer (3DHistech, Hungary.) These images were then analyzed using ImageJ/FIJI

(NIH, USA, https://imagej.net/Citing) to count the nuclei after applying a median filter,

background subtraction, and finding maxima for each nucleus.

Nitrite level determination by colorimetric assays

The NO colorimetric assay was used according to manufacturer instructions and samples

absorbance was measured at 540 nm with wavelength correction at 690 nm. DAF-2 was

used as previously described by Eyler and colleagues [33], and samples fluorescence was

measured with excitation and emission wavelengths of 488 and 525 nm, respectively. In

both assays, the levels of NO were normalized to cell numbers.

NFĸB assays

At 24 h after transfection, FC1245 cells were cultured with CM from CAFs for an

additional 24 h. The dual luciferase assay was then performed using the Dual-Luciferase

Reporter Assay System (Promega, E1910). Normalized luciferase activity for NFĸB was

obtained by subtracting background luminescence. We then calculated the ratio of firefly

luminescence from the NFĸB reporter to Renilla luminescence from the control Renilla

luciferase vector.

RNA analysis

Cells were lysed in TRIzol reagents (Life technologies) and frozen at −80°C for future

use. Process of total RNA extraction was performed according to the manufacturer’s

instructions (Life technologies). Reverse transcription to cDNA was performed with 1.5

μg using a High Capacity cDNA Archive Kit (Life technologies). cDNA samples were

pre-amplified using the PreAmp master mix (Fluidigm) and the mixtures of all the

Taqman assays (Life technologies) to prepare pre-amplified cDNA by PCR (18 cycles),

following the manufacturer’s instruction. Pre-amplified samples were loaded in a 48.48

dynamic array integrated fluidic circuit (IFC; Fluidigm) using an MX IFC controller

(Fluidigm). mRNA expression data were processed by BioMark HD System and data

analyzed using the real-time PCR analysis software (Fluidigm). mRNA levels were

relative to 18S (ΔCt = Ct gene of interest – Ct 18S) and normalized versus the mean of

control.

Murine orthotopic PDAC xenograft model

PDAC orthotopic models were developed by injection of FC1245luc+ cells into the

parenchyma of the pancreas of C57BL/6 (B6) mice. The mice were purchased from

Jackson Laboratories.

Mice were anesthetized by inhalation of 1-2% isofluorane (Baxter Healthcare) in an

oxygen gas mixture and kept on a heated platform during surgery procedures. Meloxicam

(2.0 mg kg-1 of mice) was administered subcutaneously during the perioperative period. A

solution of bupivacaine 0.25% was administrated by intradermal injection in the area

surrounding the incision line. The abdomen was shaved and swabbed with sterile 70%

ethanol alcohol pads followed by povidone-iodine (Betadine) scrub. A small longitudinal

incision (0.5–1 cm in length) was performed in the skin and peritoneum; the spleen was

identified and gently retracted to expose the pancreas. FC1245luc+ cells 25,000 in 30 μl

containing 1:1 (v/v) cell media and basement membrane (BD Matrigel, BD Biosciences)

were injected into the parenchyma of the pancreas. The injection site was gently pressed

with a sterile cotton swab to prevent cell leakage. The pancreas and spleen were replaced

in the abdomen. The abdominal and skin incisions were closed with 4-0 Vicryl sutures

(Fischer Scientific) and sterile 9 mm wound clips, respectively. Mice were injected with a

warmed saline solution (10 ml kg-1 of mice) to facilitate rehydration and recovery from

anesthesia. Buprenorphine 0.25% was administered before recovery and dosages of 10

µL were repeated (total of 100 µL) in post-surgery, as needed every 4–6 h. The mice

were monitored for any signs of pain and distress, and tumor growth was monitored by

bioluminescence. Bioluminescence imaging was performed after intraperitoneal injection

of D-Luciferin Firely (Caliper Life Sciences, 150 mg kg-1 of mice) in a IVIS Lumina III

imaging system (Xenogen). Data were analyzed using Living Image v4.3.1 software.

Image guided X-ray radiotherapy of pancreatic tumors

Therapeutic radiation was performed using the small animal micro-irradiator

(XRad225Cx, Precision X-Ray, North Brantford, CT) with a tube voltage of 225 kVp and

13 mA. Mice were kept in anesthesia during therapy. Therapeutic radiation was delivered

in a continuous Arc using the rotatable gantry of the micro-irradiator at a single dose of

12 Gy or three doses of 6 Gy (administrated with a 48 h interval). Radiation therapy was

delivered using a circular collimator of 1 cm diameter. The 12 Gy × 1 RT and 6 Gy × 3

protocols used in this work were based on previous studies demonstrating that RT

reduces survival time of mice with PDACs [11] and that iNOS inhibition combined with

RT is effective against squamous carcinoma xenografts [65].

Western Blot

Whole-protein extracts from cells or tumors were prepared after cell scrapping or tissue

homogenization, respectively, in RIPA buffer. After centrifugation at 16,000 × g for 10

min at 4 °C, supernatants were used for protein quantification as determined with the

Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), followed by denaturation of

the sample with Laemmli buffer. Following electrophoresis and transfer to

polyvinylidene difluoride (PVDF) membranes (Bio-Rad), the blots were incubated in 5%

(m/v) BSA in Tris-buffered saline buffer-Tween (TBS-T, Cell Signaling Technology)

and probed with mouse anti-β-actin 1:20,000 (Sigma, A1978), rabbit anti-iNOS 1:500

(Abcam, ab3523) or rabbit anti-GLUT1 1∶1,000 (Sigma, 07-1401). After washing with

TBS-T, the membranes were incubated with IRDye®800CW anti-Rabbit or anti-Mouse

IgG 1:15,000 (LI-COR Biosciences) and imaged on the Odyssey Infrared Imaging

System (LI-COR Biosciences) followed by densitometric analysis.

Immunofluorescence

Human or murine pancreatic tumor samples were cryosectioned, fixed with 4% PFA in

PBS for 30 min at room temperature, and permeabilized with 0.1% Triton X-100 (Sigma-

Aldrich) in PBS containing 10% normal goat serum (Thermo Fischer Scientific) for 30

min. Samples were then washed with PBS and incubated with the indicated antibodies in

PBS containing 10% normal goat serum as described below. The following antibodies

were used for immunofluorescence: rabbit anti-iNOS 1:100 (Abcam, ab3523), rabbit anti-

alpha smooth muscle actin 1:100 (Abcam, ab5694), rabbit anti-GLUT1 1:100 (Sigma, 07-

1401), rabbit anti-sodium/hydrogen exchanger 1/NHE-1 antibody 1:100 (Abcam,

ab67314), mouse anti-cytokeratin 1:10 (Abcam, ab27988), mouse anti-GLUT1 1:100

(Abcam, ab40084), mouse anti-iNOS 1:100 (Abcam, ab49999). Sections were incubated

in a humidified chamber with the primary antibodies overnight at 4 °C, and visualized

with Alexa568 or Alexa488-conjugated secondary antibodies per the manufacturer’s

instructions (Thermo Fisher Scientific). Sections were counterstained for 10 min at room

temperature with DAPI in PBS.

Whole slide digital images were generated on Pannoramic MIDI scanner (3DHistech,

Hungary) at a resolution of 0.3250 μm per pixel. Staining area quantification was

performed with the Halo 2.3.2089.23 software with Area Quantification FL 1.2 and

Tissue Classifier 2.3 modules. The region of interest (ROI) was manually defined as

viable tumor tissue excluding necrotic tumor tissue and normal tissues surrounding the

tumor, and blood vessels containing autofluorescent red blood cells were then excluded

from the ROI using Tissue Classifier. The positive area for each marker and for

colocalization of both markers was measured.

Immunohistochemistry

For quantification of intratumoral collagen, sections were stained with Masson trichrome

and whole slide digital images were generated on a scanner (Pannoramic 250 Flash III,

3DHistech, 20x/0.8NA objective, Budapest, Hungary) at a resolution of 0.2431 μm per

pixel. Area quantification was performed with the QuPath 0.1.3 software. The ROI was

manually defined as previously described. The positive area, defined as the ratio of blue

pixels to total ROI pixels, was measured using the positive pixel count algorithm.

Qualitative examination of slides, manual ROI selection, determination of stain vectors

for Masson trichrome, and optimization and validation or algorithms, were performed by

a board-certified veterinary pathologist (SM).

Supplementary Figures

Supplementary Figure 1. Confocal images of immunofluorescence staining of iNOS

(green) in non-tumor and PDAC tumors obtained before treatment (treatment naïve, non-

RT) and after RT. DAPI (blue) was used to stain cell nuclei. Scale bars: 50 μm. Notes:

Quantification of iNOS immunofluorescence in whole scans are shown in Figure 1A of

the main manuscript and patient’s RT irradiation regimens are described in

Supplementary Table 1.

Supplementary Figure 2. Western blot of iNOS in the total lysates of FC1245

orthotopic PDAC tumors obtained 2 days post-RT therapy. Density of western blot bands

was quantified by scanning densitometry with ImageJ software. iNOS protein levels in

FC1245 PDAC tumors increase after RT (13.2 ± 0.1, mean ± S.E.M, n = 4). *P < 0.05

compared with non-RT tumors and based on a Student’s t-test.

Supplementary Figure 3. Immunohistochemistry of trichrome in FC1245 orthotopic

PDAC tumors obtained at 2 days post-RT therapy. The percentage of collagen-positive

area was similar in non-RT (10.4 ± 3.6, mean ± S.E.M, n = 3) and RT (11.4 ± 0.9, mean

± S.E.M, n = 3) tumor sections.

Supplementary Figure 4. a) Bright-field microscopy images demonstrating CAFs

isolation and expansion from FC1245 orthotopic PDAC tumors. Murine PDACs were cut

into small pieces and fibroblasts migration from the tumor tissue was induced in DME

medium containing 1 g L-1 glucose, 10 mM HEPES, 100 μg mL-1 gentamicin, 1% L-

glutamine, 100 μg ml-1 Penicillin/Streptomycin, and 20% FCS. b) Fluorescence images of

immunofluorescence staining of vimentin and α-SMA (red) in CAFs isolated from

PDACs. DAPI (blue) was used to stain cell nuclei.

Supplementary Figure 5. FC1245 were cultured with CM from CAFs or co-cultured

with CAFs using a transwell in vitro system. To mimic the high PDAC desmoplastic

stromal environment, a 1:2 tumor cell-CAF ratio was used in the co-culture experiments.

At 24 h after cells co-culture or treatment with CM from CAFs, cells were irradiated at a

dose rate of 5 Gy per minute. Supernatants or cell extracts were then analyzed after an

additional 24 h co-culture.

Supplementary Figure 6. Cell growth of FC1245 cultured in the presence or absence of

conditioned media from irradiated CAFs or co-cultured with irradiated CAFs. &P < 0.05

compared with FC1245, *P < 0.05 compared with FC1245 plus CM of RT-CAFs and

based on a Student’s t-test.

Supplementary Figure 7. NO2- levels in supernatants of SUIT-2, CAPAN-2, MIA PaCa-

2, BxPC-3 human pancreatic cancer cell lines in the presence and absence of supernatants

from human CAFs and treated with 10 μM iNOS inhibitor 1400W. Treatment of

pancreatic cancer cells, that receive an extra in vitro RT dose of 5 Gy, with conditioned

medium of CAFs increases NO2- secretion by the tumor cells. The 1400W inhibitor

decreases NO-induced by CAFs in the tumor cells. *P < 0.05, **P < 0.01, ***P < 0.001

compared with non-RT pancreatic cancer cells and based on a Student’s t-test. $$P < 0.01

compared with respective non-RT control experiment. &P < 0.05, &&P < 0.01 compared

with RT-treated cells in the presence of CM CAFs and based on a Student’s t-test.

Supplementary Figure 8. IL-6, LIF, RANTES, and IL-13 concentrations in the

supernatants of FC1245 PDAC cells, FC1245 incubated with conditioned medium from

non-RT CAFs, and FC1245 incubated with conditioned medium from RT CAFs. Cell

supernatants were collected at 24 h after cell culture and cytokines activity was

determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead 32

Plex Panel. Data are presented as mean ± S.E.M., n = 3 to 6 independent experiments. *P 

< 0.05, **P < 0.01 compared with FC1245 and based on a Student’s t-test. IL-6

interleukin-6, LIF leukemia inhibitory factor, RANTES regulated on activation normal T

cell expressed and secreted, and IL-3 interleukin-13.

Supplementary Figure 9. Gene expression heatmap of non-RT and RT fibroblasts.

Heatmap values are fold induction values normalized to non-RT fibroblasts (dark blue,

high expression and white, low expression). *Indicates statistical significance. RANTES

Regulated on Activation Normal T cell Expressed and Secreted, CXCL10 C-X-C motif

chemokine 10, CXCL9 C-X-C motif chemokine 9, IFNα4 Interferon alpha-4, IFNαR1

Interferon alpha receptor subunit 1, IFNαR2 Interferon alpha receptor subunit 2, IFN-β

Interferon-β, IFNγR1 Interferon gamma receptor 1, IL12 interleukin 12, IL1α interleukin

1 alpha, IL1 β interleukin 1 beta, IL1R1 interleukin 1 receptor type I, IL6 interleukin 6,

IL6Rα interleukin 6 receptor alpha, IRF7 interferon regulatory factor 7, NOS2 inducible

nitric oxide synthase, PDL1 programmed death-ligand 1, STAT1 Signal transducer and

activator of transcription 1, TNFα tumor necrosis factor-α, VEGF Vascular endothelial

growth factor.

Supplementary Figure 10. Confocal images and quantification (mean ± S.E.M, n = 3) of

immunofluorescence staining of α-SMA (green) in FC1245 orthotopic PDAC tumors at

14 days after cells’ implantation. Pancreatic orthotopic tumors were developed by

implantation of FC1245luc+ alone, FC1245luc+ mixed with non-RT CAFs, and

FC1245luc+ mixed with RT-CAFs into the pancreas of C57BL/6 (B6) mice. DAPI (blue)

was used to stain cell nuclei. Scale bars: 50 μm.

Supplementary Figure 11. FC1245 migration images and quantification using the

wound healing assay. FC1245 PDAC cells cultured for 24 h in the presence or absence of

conditioned medium from CAFs obtained from non-RT and RT-treated mice were

wounded by scratch injury (black lines). The wound closure was measured at 4, 24, and

48 h after cell scratching. Data are presented as mean ± S.E.M, n = 3. **P < 0.01 compared

with FC1245 and based on a Student’s t-test.

Supplementary Figure 12. Liver and pancreatic tumors at 6 days after surgery.

Pancreatic orthotopic tumors were developed by implantation of FC1245luc+ alone,

FC1245luc+ mixed with non-RT CAFs, and FC1245luc+ mixed with RT-CAFs into the

pancreas of C57BL/6 (B6) mice. L liver, P pancreatic tumors.

Supplementary Figure 13. Liver, stomach, spleen, and pancreatic tumors at 14 days

after surgery. Pancreatic orthotopic tumors were developed by implantation of

FC1245luc+ alone, FC1245luc+ mixed with non-RT CAFs, and FC1245luc+ mixed with

RT-CAFs into the pancreas of C57BL/6 (B6) mice. L liver, P pancreatic tumors, St

stomach, Sp spleen.

Supplementary Figure 14. FC1245 cell growth and NO2- levels in supernatants of

FC1245 with or without 1 μM DETA-NONOate for 6 h, 10 μM of 1400W or with the

combination of DETA-NONOate/1400W. Data are presented as mean ± S.E.M, n = 4. *P 

< 0.05, ***P < 0.001 compared with FC1245 and based on a Student’s t-test. &P < 0.05,

&&P < 0.01 compared with DETA-NO and based on a Student’s t-test. DETA-NO NO

donor diethylenetriamine NONOate, 1400W N-(3-(aminomethyl)benzyl)acetamidine.

Supplementary Figure 15. FC1245 cell growth and NO2- levels in supernatants of

FC1245 transfected with iNOS CRISPR activation plasmid or control CRISPR activation

plasmid. Data are presented as mean ± S.E.M, n = 4. *P < 0.05, ***P < 0.001 compared

with control CRISPR activation plasmid and based on a Student’s t-test. &P < 0.05, &&P < 

0.01 compared with iNOS CRISPR activation plasmid and based on a Student’s t-test.

Supplementary Figure 16. Cell growth of FC1245 cultured in the presence or absence

of irradiated and non-irradiated CAFs. To deplete iNOS, FC1245 tumor cells were

transfected with iNOS siRNA (iNOS KD) or control scrambled siRNA (Scr). Data are

presented as mean ± S.E.M., n = 3. *P < 0.05 compared with FC1245, &P < 0.05

compared with FC1245 plus respective CAFs.

Supplementary Figure 17. Survival of mice implanted with FC1245 orthotopic PDAC

tumors and treated with 12 Gy in a single treatment fraction or 18 Gy administered in

three fractions at 48 h intervals.

Supplementary Figure 18. Tumor weight in PDAC tumors from RT, 1400W, and

1400W/RT treated mice (n = 10 mice per group) on day 14 post-therapy. Mice were

treated with 3 doses of 6 Gy at 48 h intervals. Intraperitoneal administration of 1400W (1

μg g-1) was started at day 0. 1400W was administrated every day for 7 days during the RT

treatment period and every two days during two weeks after the RT treatment period. **P 

< 0.01 compared with RT treated group and based on a Student’s t-test. &P < 0.01

compared with 1400W treated group and based on a Student’s t-test.

Supplementary Figure 19. Glucose uptake and lactate production of FC1245 PDAC

cells treated with or without 1 μM NO donor DETA-NO, 10 μM iNOS inhibitor 1400W,

and the combination of DETA-NO/1400W. Glucose consumption and lactate production

increase after incubation with DETA-NO, an effect that is inhibited by the addition of the

iNOS inhibitor 1400W.

Supplementary Figure 20. Biodistribution data at 2 h after injection of 18F-FDG in mice

bearing FC1245 orthotopic tumors. At 14 days post-therapy, mice were administrated

200 μCi intravenous injection of 18F-FDG. Bars, n = 4 mice per group, mean ± S.E.M.

%ID, percentage of injected dose.

Supplementary Figure 21. Biodistribution data at 24 h after injection of 67Ga-labeled

pHLIP in mice bearing FC1245 orthotopic tumors. At 14 days post-therapy, mice were

administrated 20 μCi intravenous injection of 67Ga-labeled pHLIP. Bars, n = 4 mice per

group, mean ± S.E.M. %ID g−1, percentage of injected dose per gram.

Supplementary Tables

Supplementary Table 1. RT administrations, time-points, and human PDAC sample collection time-points.

Patient # RSX # RT doses given (mGy-cm)

RT data given

Months before sample collection

Procedure collection date

# Fractionation doses

Cumulative dose

1 RSX295 1734 5/24/16 ~ 10 3/13/17 7 101392006 8/11/16 ~ 71661 10/15/16 ~ 5617 12/21/16 ~ 31899 1/5/17 ~ 21206 1/26/17 ~ 21016 2/21/17 ~ 0.6

2 RSX44 2245 3/31/15 ~ 5 8/24/15 3 68062212 6/19/15 ~ 22349 7/31/15 ~ 0.2

3 RSX199 1202 1/23/16 ~ 8 9/14/16 5 66991877 3/30/16 ~ 61079 5/25/16 ~ 41904 7/8/16 ~ 2637 8/25/16 ~ 0.6

4 RSX349 1601 10/26/16 ~ 8 6/21/17 4 61041734 2/6/17 ~ 41335 4/7/17 ~ 21434 5/30/17 ~ 0.7

Supplementary Table 2. Quantification of iNOS+/α-SMA+ in PDAC RT-treated samples obtained from patient #3. Representative fluorescence images are shown in Fig. 1B.Number of cells (DAPI/α-SMA) 18000 20500 19000 14500Number of cells (DAPI/iNOS/α-SMA) 10000 17600 10200 4500%cells iNOS+/α-SMA+ 55.6 85.9 53.7 31.0Mean 56.5S.D. 22.5N 4.0

Supplementary Table 3. Quantification of iNOS+/cytokeratin+ in PDAC RT-treated samples obtained from patient #3. Representative fluorescence images are shown in Fig. 1C.Number of cells (DAPI/cytokeratin) 30400 22500 29000 20300Number of cells (DAPI/iNOS/cytokeratin) 28900 21200 26000 16500%cells iNOS+/cytokeratin+ 95.1 94.2 89.7 81.3Mean 90.1S.D. 6.3N 4.0

Supplementary Table 4. Quantification of iNOS+/α-SMA+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Representative fluorescence images are shown in Fig. 1E.Number of cells (DAPI/α-SMA) 243923 223124 213413 203113Number of cells (DAPI/iNOS/α-SMA) 1133 1021 1220 1002%cells iNOS+/α-SMA+ 0.46 0.46 0.57 0.49Mean 0.46S.D. 0.05N 4.0

Supplementary Table 5. Quantification of iNOS+/α-SMA+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Representative fluorescence images are shown in Fig. 1E.Number of cells (DAPI/α-SMA) 530482 503241 450234 402412Number of cells (DAPI/iNOS/α-SMA) 4347 4214 3890 3217%cells iNOS+/α-SMA+ 0.82 0.84 0.86 0.80Mean 0.82S.D. 0.03N 4.0

Supplementary Table 6. Quantification of iNOS+/cytokeratin+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Representative fluorescence images are shown in Fig. 1E.Number of cells (DAPI/cytokeratin) 395208 298353 389274 283475Number of cells (DAPI/iNOS/cytokeratin) 146382 73482 74526 38082%cells iNOS+/cytokeratin+ 37.0 24.6 19.1 13.4Mean 23.6S.D. 10.1N 4.0

Supplementary Table 7. Quantification of iNOS+/cytokeratin+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Representative fluorescence images are shown in Fig. 1E.Number of cells (DAPI/cytokeratin) 133428 132324 122383 133228Number of cells (DAPI/iNOS/cytokeratin) 74208 134212 134224 123242%cells iNOS+/cytokeratin+ 55.6 101.4 109.7 92.5Mean 89.8S.D. 23.8N 4.0

Supplementary Table 8. Quantification of GLUT1+/α-SMA+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Representative fluorescence images are shown in Fig. 4C.Number of cells (DAPI/α-SMA) 75200 56213 80231 70131Number of cells (DAPI/GLUT1/α-SMA) 1 2 0 0%cells GLUT1+/α-SMA + 0.5 1.4 0.0 0.0Mean 0.5S.D. 0.7N 4.0

Supplementary Table 9. Quantification of GLUT1+/α-SMA+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Representative fluorescence images are shown in Fig. 4C.Number of cells (DAPI/α-SMA) 22000 15024 10324 20131Number of cells (DAPI/GLUT1/α-SMA) 200 300 0.0 0.0%cells GLUT1+/α-SMA + 0.9 2.0 0.0 0.0Mean 0.7S.D. 0.9N 0.0

Supplementary Table 10. Quantification of GLUT1+/cytokeratin+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Representative fluorescence images are shown in Fig. 4D.Number of cells (DAPI/cytokeratin) 171200 165231 121338 145221Number of cells (DAPI/GLUT1/cytokeratin) 10000 8000 5021 4523%cells GLUT1+/cytokeratin+ 5.8 4.8 4.1 3.1Mean 4.5S.D. 1.1N 4.0

Supplementary Table 11. Quantification of GLUT1+/cytokeratin+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Representative fluorescence images are shown in Fig. 4D.Number of cells (DAPI/cytokeratin) 134800 143923 153923 170342Number of cells (DAPI/GLUT1/cytokeratin) 41600 56024 49242 60321%cells GLUT1+/cytokeratin+ 30.9 38.9 32.0 35.4Mean 34.3S.D. 3.6N 4.0

References

1. Ohuchida, K.; Mizumoto, K.; Murakami, M., et al. Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interactions. Cancer Res. 2004, 64, 3215-3222.2. Cardnell, R. J.; Mikkelsen, R. B. Nitric oxide synthase inhibition enhances the antitumor effect of radiation in the treatment of squamous carcinoma xenografts. Plos One. 2011, 6, e20147.3. Caneba, C. A.; Yang, L.; Baddour, J., et al. Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells. Cell Death Dis. 2014, 5.