iNOS Regulates the Therapeutic Response of …...Pancreatic ductal adenocarcinoma (PDAC) accounts...

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CANCER RESEARCH | MOLECULAR CELL BIOLOGY iNOS Regulates the Therapeutic Response of Pancreatic Cancer Cells to Radiotherapy A C Patricia M.R. Pereira 1 , Kimberly J. Edwards 1 , Komal Mandleywala 1 , Lukas M. Carter 1 , Freddy E. Escorcia 2 , Luis Felipe Campesato 3 , Mike Cornejo 1 , Lolkje Abma 1 , Abu-Akeel Mohsen 3 , Christine A. Iacobuzio-Donahue 4,5 , Taha Merghoub 3,6 , and Jason S. Lewis 1,7,8,9,10 ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to radiotherapy, chemotherapy, or a combination of these modalities, and surgery remains the only curative intervention for localized disease. Although cancer-associated broblasts (CAF) are abundant in PDAC tumors, the effects of radiotherapy on CAFs and the response of PDAC cells to radiotherapy are unknown. Using patient samples and orthotopic PDAC biological models, we showed that radiotherapy increased inducible nitric oxide synthase (iNOS) in the tumor tissues. Mechanistic in vitro studies showed that, although undetectable in radiotherapy-activated tumor cells, iNOS expres- sion and nitric oxide (NO) secretion were signicantly increased in CAFs secretome following radiotherapy. Culture of PDAC cells with conditioned media from radiotherapy-activated CAFs increased iNOS/NO signaling in tumor cells through NF-kB, which, in turn, elevated the release of inammatory cytokines by the tumor cells. Increased NO after radiotherapy in PDAC contributed to an acidic microenvironment that was detectable using the radiolabeled pH (low) insertion peptide (pHLIP). In murine orthotopic PDAC models, pancreatic tumor growth was delayed when iNOS inhibi- tion was combined with radiotherapy. These data show the impor- tant role that iNOS/NO signaling plays in the effectiveness of radiotherapy to treat PDAC tumors. Signicance: A radiolabeled pH-targeted peptide can be used as a PET imaging tool to assess therapy response within PDAC and blocking iNOS/NO signaling may improve radiotherapy outcomes. Introduction Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all the pancreatic cancer cases and it remains one of the most lethal malignancies due to limited treatment options and resistance to therapy (1). Notably, limited improvements in overall survival have been achieved for PDAC in comparison with other tumor types, in part, because more than half of PDAC cases are diagnosed at an advanced and metastatic stage of the disease (1). Early detection of PDAC remains a challenge due to nonspecic presenting symp- toms, difculty of imaging early-stage tumors, and lack of tumor biomarkers with both good specicity and sensitivity. The poor prognosis for PDAC can be attributed to the rapid metastasis and resistance to conventional therapeutic approaches (chemotherapy, radiotherapy, and molecular targeted therapy). Clearly, a need exists to identify new therapeutic strategies, including effective combina- tions of existing therapies to expand treatment options for PDAC. Emerging therapeutic approaches for PDAC include immuno- therapies, molecular targeted therapies, and strategies targeting the tumor microenvironment (2). While pancreaticoduodenectomy (Whipple procedure) or distal pancreatectomy is performed in patients with resectable PDAC, fewer than 20% of all patients fall into these categories, and only cures about 10% of this subset (3). Patients with unresectable PDAC are treated with chemotherapy or chemotherapy/radiation combi- nation therapies (4). Technological improvements in radiotherapy (RT) approaches, such as intensity-modulated radiotherapy, and image-guided radiotherapy, have dramatically improved the conformality of the radiation dose deposition in the tumor area while increasingly sparing healthy tissues (5). Radiotherapy induces alterations in the secretory prole of tumor microenvironment (TME) that results in changes in tumor invasion, tumor growth, inammatory mediators, and regulators of angiogen- esis (68). Noncancer cells including immune cells, broblasts, peri- cytes, and endothelial cells contribute to the stromal compartment of the TME, and, in PDAC, outnumbers the cancer cells (2, 9). The TME of PDAC is highly inammatory (10); radiotherapy alters the inam- matory TME by contributing to a broinammatory desmoplasia (11) and to an increase in the expression of TNFa (12). The proinam- matory cytokines IFNa, IFNb, and IFNg are further induced in radiotherapy-treated cells (13, 14). Cancer-associated broblasts (CAF) expressing both vimentin and a-smooth muscle actin (aSMA) are the predominant cellular components of the PDAC stroma (2). Previous studies have demonstrated that irradiated pancreatic CAFs 1 Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York. 2 Molecular Imaging Program, Center for Cancer Research, NCI, NIH, Bethesda, Maryland. 3 Swim Across America and Ludwig Collaborative Labora- tory, Immunology Program, Parker Institute for Cancer Immunotherapy, Memo- rial Sloan Kettering Cancer Center, New York, New York. 4 The David M. Rubenstein Center for Pancreatic Cancer Research, Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York. 5 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York. 6 Department of Medicine, Weill Cornell Medical College, New York, New York. 7 Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, New York. 8 Department of Pharmacology, Weill Cornell Medical College, New York, New York. 9 Department of Radiology, Weill Cornell Medical College, New York, New York. 10 Radiochemistry and Molecular Imaging Probes Core, Memorial Sloan Kettering Cancer Center, New York, New York. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Jason S. Lewis, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, NY 10065. Phone: 646-888-3038; Fax: 646-888- 3059; E-mail: [email protected] Cancer Res 2020;80:168192 doi: 10.1158/0008-5472.CAN-19-2991 Ó2020 American Association for Cancer Research. AACRJournals.org | 1681 on June 11, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst February 21, 2020; DOI: 10.1158/0008-5472.CAN-19-2991
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  • CANCER RESEARCH | MOLECULAR CELL BIOLOGY

    iNOS Regulates the Therapeutic Response of PancreaticCancer Cells to Radiotherapy A CPatricia 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, and Jason S. Lewis1,7,8,9,10

    ABSTRACT◥

    Pancreatic ductal adenocarcinoma (PDAC) is highly resistant toradiotherapy, chemotherapy, or a combination of these modalities,and surgery remains the only curative intervention for localizeddisease. Although cancer-associated fibroblasts (CAF) are abundantin PDAC tumors, the effects of radiotherapy on CAFs and theresponse of PDAC cells to radiotherapy are unknown. Using patientsamples and orthotopic PDAC biological models, we showed thatradiotherapy increased inducible nitric oxide synthase (iNOS) in thetumor tissues. Mechanistic in vitro studies showed that, althoughundetectable in radiotherapy-activated tumor cells, iNOS expres-sion and nitric oxide (NO) secretion were significantly increasedin CAFs secretome following radiotherapy. Culture of PDACcells with conditioned media from radiotherapy-activated CAFs

    increased iNOS/NO signaling in tumor cells throughNF-kB, which,in turn, elevated the release of inflammatory cytokines by the tumorcells. Increased NO after radiotherapy in PDAC contributed to anacidic microenvironment that was detectable using the radiolabeledpH (low) insertion peptide (pHLIP). In murine orthotopic PDACmodels, pancreatic tumor growth was delayed when iNOS inhibi-tion was combined with radiotherapy. These data show the impor-tant role that iNOS/NO signaling plays in the effectiveness ofradiotherapy to treat PDAC tumors.

    Significance:Aradiolabeled pH-targeted peptide can be used as aPET imaging tool to assess therapy response within PDAC andblocking iNOS/NO signaling may improve radiotherapy outcomes.

    IntroductionPancreatic ductal adenocarcinoma (PDAC) accounts for 90% of

    all the pancreatic cancer cases and it remains one of the most lethalmalignancies due to limited treatment options and resistance totherapy (1). Notably, limited improvements in overall survival havebeen achieved for PDAC in comparison with other tumor types, inpart, because more than half of PDAC cases are diagnosed at anadvanced and metastatic stage of the disease (1). Early detection ofPDAC remains a challenge due to nonspecific presenting symp-

    toms, difficulty of imaging early-stage tumors, and lack of tumorbiomarkers with both good specificity and sensitivity. The poorprognosis for PDAC can be attributed to the rapid metastasis andresistance to conventional therapeutic approaches (chemotherapy,radiotherapy, and molecular targeted therapy). Clearly, a need existsto identify new therapeutic strategies, including effective combina-tions of existing therapies to expand treatment options for PDAC.Emerging therapeutic approaches for PDAC include immuno-therapies, molecular targeted therapies, and strategies targeting thetumor microenvironment (2).

    While pancreaticoduodenectomy (Whipple procedure) or distalpancreatectomy is performed in patients with resectable PDAC,fewer than 20% of all patients fall into these categories, and onlycures about 10% of this subset (3). Patients with unresectable PDACare treated with chemotherapy or chemotherapy/radiation combi-nation therapies (4). Technological improvements in radiotherapy(RT) approaches, such as intensity-modulated radiotherapy, andimage-guided radiotherapy, have dramatically improved theconformality of the radiation dose deposition in the tumor areawhile increasingly sparing healthy tissues (5).

    Radiotherapy induces alterations in the secretory profile of tumormicroenvironment (TME) that results in changes in tumor invasion,tumor growth, inflammatory mediators, and regulators of angiogen-esis (6–8). Noncancer cells including immune cells, fibroblasts, peri-cytes, and endothelial cells contribute to the stromal compartment ofthe TME, and, in PDAC, outnumbers the cancer cells (2, 9). The TMEof PDAC is highly inflammatory (10); radiotherapy alters the inflam-matory TME by contributing to a fibroinflammatory desmoplasia (11)and to an increase in the expression of TNFa (12). The proinflam-matory cytokines IFNa, IFNb, and IFNg are further induced inradiotherapy-treated cells (13, 14). Cancer-associated fibroblasts(CAF) expressing both vimentin and a-smooth muscle actin (aSMA)are the predominant cellular components of the PDAC stroma (2).Previous studies have demonstrated that irradiated pancreatic CAFs

    1Department of Radiology, Memorial Sloan Kettering Cancer Center, New York,New York. 2Molecular Imaging Program, Center for Cancer Research, NCI, NIH,Bethesda, Maryland. 3Swim Across America and Ludwig Collaborative Labora-tory, Immunology Program, Parker Institute for Cancer Immunotherapy, Memo-rial Sloan Kettering Cancer Center, New York, New York. 4The David M.Rubenstein Center for Pancreatic Cancer Research, Human Oncology andPathogenesis Program, Memorial Sloan Kettering Cancer Center, New York,New York. 5Department of Pathology, Memorial Sloan Kettering Cancer Center,New York, New York. 6Department of Medicine, Weill Cornell Medical College,New York, New York. 7Molecular Pharmacology Program, Memorial SloanKettering Cancer Center, New York, New York. 8Department of Pharmacology,Weill Cornell Medical College, New York, New York. 9Department of Radiology,Weill Cornell Medical College, New York, New York. 10Radiochemistry andMolecular Imaging Probes Core, Memorial Sloan Kettering Cancer Center, NewYork, New York.

    Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

    Corresponding Author: Jason S. Lewis, Memorial Sloan Kettering CancerCenter, 1275 York Avenue, NY 10065. Phone: 646-888-3038; Fax: 646-888-3059; E-mail: [email protected]

    Cancer Res 2020;80:1681–92

    doi: 10.1158/0008-5472.CAN-19-2991

    �2020 American Association for Cancer Research.

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    on June 11, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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  • exhibit enhanced tumor–stroma interactions, which impact tumorgrowth and invasion (7, 15). Pancreatic CAFs survive severe stress,including damage induced by radiotherapy single-fraction doses up to20 Gy (16).

    The expression of inducible nitric oxide synthase (iNOS, NOS2)in tumor cells is associated with poor survival in several cancers(17–20). The iNOS is transcriptionally regulated and induced byinflammatory cytokines (21, 22), contributing to the production ofnitric oxide (NO) through conversion of L-arginine into citrullinein the presence of NADPH and oxygen. An increase in NO resultsin an intrinsic prooxidant environment (22), immune escape (23),and resistance to apoptosis (24). NO shows promise either as astandalone therapeutic agent or as target of cancer therapies. Therole of NO in cancer depends on the NO concentration or durationof NO exposure, extracellular conditions, localization of NOS, andcellular sensitivity to NO (21, 25–27). Pancreatic tumors exhibithigher expression of iNOS compared with nontumor pancreatictissue (28). Pancreatic orthotopic implantation of tumor cells ex-pressing low levels of iNOS result in the formation of pancreatictumors with metastasis in the liver and formation of ascites—aneffect that is not observed in tumor models developed by orthotopicimplantation of PDAC cells containing high levels of iNOS (21).In addition to or independent of iNOS expression in tumor cells,upregulation of iNOS has been detected in stromal fibroblastsand immune cells (22). Mechanistic studies have demonstratedthat in PDAC, CAFs express high amounts of iNOS contributingto the development of tumor chemoresistance via increased NOsecretion (29). Inflammatory cytokines induce NO productionby CAFs (29), contributing to PDAC chemoresistance (30) andimmunosuppression (31).

    Not only does NO contribute to tumor growth and metasta-sis (21, 26, 32, 33), but mechanistic metabolic studies demonstrateda NO-mediated decrease in mitochondrial respiration, which ledcancer cells to undergo higher glycolytic rates to maintain ATPproduction levels (34). These results have further supported the roleof NO in cancer metabolism by demonstrating that NO regulates theWarburg effect and promotes cancer growth by inhibiting mitochon-drial respiration.

    We hypothesize that radiotherapy induces alterations in theinflammatory TME contributing to an increase in PDAC iNOSexpression and NO secretion that results in an increase in tumorgrowth. In this study, we show that conditioned media (CM) fromradiotherapy-activated CAFs (RT-CAF) increase NO secretion/iNOS expression by the tumor cells that contributes to PDACtumor growth, and acidification of the PDAC microenvironment,which can be detected using the radiolabeled pH (low) insertionpeptide (pHLIP). Importantly, we demonstrate that this phenom-enon can be mitigated with iNOS inhibition in vitro and in vivo,providing a therapeutic option for overcoming this protumorigenicphenotype.

    Materials and MethodsPancreatic cancer cell lines and patient samples

    SUIT-2, CAPAN-2, MIA PaCa-2, and BxPC-3 human PDACs werepurchased from ATCC and were grown according to standard pro-cedures. The FC1245 murine PDACs were obtained from the DavidTuvenson's group and were transformed in Michel Sadelain's labora-tory to generate FC1245lucþ (35). FC1245 and FC1245lucþ cells werecultured in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin. Cell lines used in this work were purchased in 2016–

    2019, and theywere usedwithin passage number of 15. All the cell lineswereMycoplasma free and authenticated atMemorial Sloan-KetteringCancer Center (MSKCC, New York, NY) integrated genomics oper-ation core using short tandem repeat analysis.

    Deidentified patient samples (nontumor and tumor pancreatictissues) were obtained before and after radiotherapy from David M.Rubenstein Center for Pancreatic Cancer Research following Institu-tional Review Board approval. Radiotherapy doses, radiotherapytime-points, and sample collection time-points are described in Sup-plementary Table S1.

    Murine orthotopic PDAC xenograft modelAll experiments involving animals were performed according

    to the guidelines approved by the Research Animal ResourceCenter and Institutional Animal Care and Use Committee atMSKCC. PDAC orthotopic models were developed as describedpreviously (36) and additional details are given in SupplementaryInformation.

    Contrast injection and CT for pancreatic tumor delineationPancreatic tumors were visualized using an image-guided small-

    animal micro-irradiator (XRad225Cx, Precision X-Ray) following aprotocol previously described by Thorek and colleagues (37). Micewere injected with a 3 mL solution of 75 mg Iodine/mL (iohexol;Omnipaque, GE Healthcare) at 5 minutes prior to imaging. Datavisualization was performed as described previously (37) and tumorvolumes were determined using Amira region-grow tool.

    Image-guided X-ray radiotherapy of pancreatic tumorsImmediately after visualization of the pancreatic tumor byX-rayCT

    using a solution of 15 mg iodine/mL, radiation arc therapy wasperformed in mice bearing orthotopic pancreatic tumors (37). SeeSupplementary Information for details.

    Isolation and expansion of murine and human pancreaticfibroblasts

    Fibroblasts were isolated from orthotopic FC1245 PDAC modelsor human PDAC using methodology described previously byM€uerk€oster and colleagues (29). Further details are described inSupplementary Information.

    Conditioned medium from irradiated and nonirradiated cellsTo analyze the effects of radiotherapy on NO secretion from

    fibroblasts on tumor cells and vice versa, cells were cultured with therespective conditionedmediumobtained fromfibroblasts for 24 hours.Before use, these media was centrifuged at 10,000 rpm for 10 minutes.

    Coculture of irradiated and nonirradiatedpancreatic tumor cellswith fibroblasts

    FC1245 pancreatic tumor cells (1 � 105 tumor cells per well) wereplated into the bottom compartment of a 6-well culture plate (Corn-ing). The fibroblasts were then seeded into the top transwell com-partment (2 � 105 fibroblasts per well). Medium was replaced after24 hours, cocultures were irradiated (see below for details on in vitroradiotherapy treatments), and supernatants or cell extracts wereanalyzed after an additional 24-hour coculture. The supernatants werecentrifuged at 10,000 rpm for 10 minutes before use on the analysis.

    Nitrite level determination by colorimetric assaysNO is a gaseous free radical with a short life and no available

    methods to directly measure NO levels exist. Therefore, the levels of

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  • more stable NO metabolites (nitrite, NO2�) were measured in cell

    culture supernatants using the NO colorimetric assay (R&D Systems)or the fluorescent probe 4,5-Diaminofluorescein (DAF-2; CaymanChemical). See Supplementary Information for details.

    In vitro therapeutic treatmentsDetails on the different in vitro therapeutic experiments—radiation

    treatment, iNOS inhibition with 1400W, iNOS knockdown, iNOSamplification, NO donor treatments, glucose uptake, lactate produc-tion, cell counting, viability assessment with Trypan blue exclusion,migration, and invasion assays—are described in SupplementaryInformation.

    NFkB, cytokine, and RNA analysesThe activity of the NFkB in FC1245 cells cultured in the presence

    and absence of CM from CAFs was evaluated using a NFkB reportedkit according to the manufacturer's instructions (Bioscience, 60614).See Supplementary Information for details

    Cell supernatants of FC1245 PDAC cells, radiotherapy fibroblasts,nonradiotherapy fibroblasts, and FC1245 incubated with CM fromfibroblastswere collected, centrifuged at 16,000� g for 10minutes, andsaved at �80 �C until the day of the experiment. Cytokines werequantified using the MILLIPLEX MAP Mouse Cytokine/ChemokineMagnetic Bead 32 Plex Panel (Millipore).

    For RNA analyses, details are described in Supplementary Infor-mation. mRNA expression data were processed by BioMark HDSystem and data analyzed using the real-time PCR analysis software(Fluidigm).mRNA levels were relative to 18S (DCt¼Ct gene of interest– Ct 18S) and normalized versus the mean of control.

    In vivo therapy studiesBioluminescencewasmeasured for 5minutes after intraperitoneally

    administering 150 mg/kg of potassium D-luciferin salt (Caliper LifeSciences) dissolved in PBS. Mice were deemed to have engraftedtumors (typically 7 days postorthotopic implantation of FC1245PDAC cells) once total bioluminescence surpassed 105 photons/second/cm2/sr. Mice were randomly grouped into treatment groups(n¼10per group): vehicle, 1400W, radiotherapy, or a combinationof1400Wwith radiotherapy. Radiotherapy groups received three dosesof 6 Gy (administered with a 48-hour interval, see sections above fordetails on radiotherapy protocols). Intraperitoneal administration of1400W (1 mg/g) was started at day 0. 1400W was administeredonce daily for 7 days during the radiotherapy treatment period andevery 2 days for 2 weeks after the radiotherapy treatment period.

    Western blotting, immunofluorescence, and IHCWestern blot, immunofluorescence, and IHC were performed as

    detailed in Supplementary Information.

    Radiolabeling of pHLIP, PET imaging of 18F-FDG, and acutebiodistribution studies

    The 18F-FDG was obtained from the Nuclear Pharmacy at MSKCCon the morning of injection.

    NO2A-variant 3 pHLIP (>95% chemical purity) was purchased andused as received from CSBio Co. For peptide radiolabeling withGallium-67 (67Ga), 67Ga (gallium-67 citrate, Nuclear Diagnostic Pro-ducts) was trapped on a silica cartridge (Sep-Pak Light, Waters),washed with water, and eluted as [67Ga]GaCl3 with 0.4 mol/L HCl,followed by pH adjustment to approximately 4.0 with 1.0 mol/LNa2CO3. NO2A-cysVar3 was dissolved in DMSO and added to the[67Ga]GaCl3. Following addition of 200 mL of AcN, the mixture was

    incubated at approximately 75�C for 30 minutes, diluted with water,and loaded onto a C-18 cartridge (Sep-Pak Light, Waters). Afterwashing the cartridge with 10 mL of water to remove unbound 67Ga,the radiolabeled peptide was eluted with 100% ethanol and recon-stituted in PBS for in vivo studies. The radiochemical purity of allconstructs used in animal studies exceeded 95%.

    At 14 days posttherapy (see above for details on the radiotherapyprotocol and 1400W administrations), mice (n ¼ 5 per group) wereadministered 20 mCi intravenous injection of 67Ga-labeled pHLIP or200 mCi 18F-FDG. At 2 hours postintravenous administration of 18F-FDG, PET images were recorded on an Inveon PET scanner (Siemens).All images were visualized in AMIDE 1.0.4 software (http://amide.sourceforge.net).

    Acute biodistribution studies were performed at 24 hours afterinjection of 67Ga-labeled pHLIP or 2 hours after intravenous injectionof 18F-FDG.Mice were sacrificed and organs were harvested, weighed,and assayed in the gamma counter for biodistribution studies. Radio-activity associated with each organ was expressed as percentage ofinjected dose per gram of organ (%ID/g).

    ResultsRadiotherapy increases iNOS expression in murine and humanpancreatic tumors

    Given that a higher iNOS expression is associatedwith poor survivalin patients with PDAC (20) and with PDAC therapeutic resis-tance (29), we sought to determine whether radiotherapy interfereswith iNOS protein levels in pancreatic cancer. We used immunoflu-orescence assays to characterize iNOS protein levels in matchednormal versus tumor pancreatic samples from patient specimensobtained before treatment [treatment-na€�ve, nonradiotherapy (non-RT)] and after radiotherapy (Fig. 1A; Supplementary Fig. S1). Theradiotherapy-treated PDAC samples were obtained between 1 and3 weeks after the last radiotherapy treatment fraction (SupplementaryTable S1) and the number of cumulative radiation dose given to thosepatients decreased in the following order patient #1 > patient #2 >patient #3 > patient #4. The tumor tissues in our four different patientcohorts showed a significantly higher iNOS expression when com-pared with the nontumor pancreas (Fig. 1A; Supplementary Fig. S1).These observations are consistent with previous IHC staining showinghigher iNOS expression in tumor when compared with nontumorsamples in PDAC cases (20, 28). The iNOS protein levels were higherin radiotherapy-treated tumors when compared with nontreatedPDAC tumors (4.6-fold � 0.1 patient #1, 1.6-fold � 0.4 patient #2,2.4-fold � 0.9 patient #3, and 1.8-fold � 0.5 patient #4; Fig. 1A).Immunofluorescence staining of iNOSwas similar in nonradiotherapyversus radiotherapy-treated nontumor tissues of patients #2 and #3.Nontumor samples of 2 other patients showed a 2-fold increase iniNOS protein levels after radiotherapy (2.4� 0.3 patient #1 and 2.3�0.5 patient #4). Additional immunofluorescence staining ofaSMAandcytokeratin in radiotherapy-treated samples obtained from patient #3,demonstrated iNOSþ/aSMAþ in 56.5% of total aSMAþ cells andiNOSþ/cytokeratinþ in 90.1% of total cytokeratinþ cells (Fig. 1Band C; Supplementary Tables S2 and S3).

    To further compare iNOS protein levels between nonradiotherapyand radiotherapy-treated pancreatic tumors, FC1245lucþ murinepancreatic cancer cells were orthotopically transplanted to the pan-creas of C57BL/6 (B6) mice. FC1245 pancreatic cancer cells isolatedfrom KPC (KrasLSL-G12D/þ;Trp53LSL-R172H/þ;Pdx1-Cre) mice in theC57BL/6 (B6) genetic background represent a valuable biologicalPDAC model (38) as they mimic the pathophysiologic features of

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  • human PDAC, and when transplanted intomice they develop invasivemetastatic PDAC characterized by extensive stroma (39). Murineradiotherapy treatment planning utilized CT imaging with intraper-itoneally administered contrast agent to define the orthotopic PDACtumor area and the entire tumor mass was selectively irradiated with asingle radiotherapy therapeutic absorbed dose of 12 Gy using an arctreatment (37). The use of a 12 Gy dose to study radiotherapy-mediated iNOS production was based on previous studies demon-strating that radiotherapy at this dose accelerates the progression ofPDAC invasion (11), which we hypothesize will correspondinglyeffectuate radiotherapy-mediated iNOS production. Western blot(Supplementary Fig. S2) and immunofluorescence (Fig. 1D) analysesof murine orthotopic PDACs obtained at 2 days after radiotherapyconfirms the increase in iNOS protein levels (13.2-fold� 0.1, mean�SEM, n ¼ 4). Radiotherapy-treated PDAC exhibited a 5-fold increasein SMA when compared with non-radiotherapy PDAC (Fig. 1D;Supplementary Tables S4 and S5). Quantification of iNOS colocaliza-tion with aSMA and cytokeratin in FC1245 radiotherapy-treatedtumors demonstrated a 2-fold increase iNOSþ/aSMAþ and a 5-foldincrease iNOSþ/cytokeratinþ when compared with non-radiotherapyPDAC (Fig. 1D and E; Supplementary Tables S4–S7). Additional IHCstudies demonstrated that under the conditions of our experiments,the percentage of collagen-positive cells is similar in nonradiotherapy(10.4 � 3.6) and radiotherapy (11.4 � 0.9) FC1245 PDAC tumors(Supplementary Fig. S3). Others have also demonstrated that p48Cre;LSL-KrasG12D (KC)mice irradiated at 12Gy exhibitfibroinflammatorydesmoplasia associated with an increase in collagen and aSMA (11),suggesting pancreatic stellate cell activation. Together, these resultssuggest that local tumor irradiation induces iNOS expression inpancreatic tumors.

    Radiotherapy-activated fibroblasts induce inflammatorycytokines secretion, iNOS expression, and NO release by PDAC

    Pancreatic TME largely consists of stromal fibroblasts, which arethought to contribute to PDAC resistance to treatment (40, 41).Therefore, we examined the role of CAFs in iNOS expression andNO secretion of PDAC following radiotherapy. Previous studies havedemonstrated increased invasiveness of pancreatic cancer cells aftercoculture with fibroblasts irradiated at a dose of 5 Gy (7). Weperformed primary cultures of fibroblasts (29) isolated from surgicalresections of treatment na€�ve (nonradiotherapy) and radiotherapy-treated FC1245 PDACs (Supplementary Fig. S4A and S4B). Toinvestigate possible paracrine iNOS/NO signaling, we analyzed CMof radiotherapy versus nonradiotherapy CAFs and in vitro coculturemodels consisting of FC1245 pancreatic cancer cells and CAFs(Fig. 2A; Supplementary Fig. S5). To mimic the high PDAC desmo-plastic stromal environment, we followed previous reports and used atumor cell–CAF ratio of 1:2 in our in vitromodel system (29).Westernblot analysis demonstrated similar iNOS expression in FC1245 cancercells andCAFs (Fig. 2B). Additional studies in FC1245 cancer cells andCAFs that received an extra in vitro irradiation dose of 5 Gy demon-strated, respectively, a 2.2-fold and 3.3-fold increase in iNOS proteinlevels in non-RT CAFs and RT-CAFs (Fig. 2B). iNOS expression wassimilar in nonradiotherapy and radiotherapy-treated FC1245 cells(Fig. 2B). Additional Western blot analysis revealed a 1.4-fold �0.1 increase in iNOS expression when FC1245 cancer cells are incu-batedwithCMofCAFs for 24 hours (Fig. 2C). Because an extra in vitroirradiation dose increases iNOS expression in CAFs (Fig. 2B), weperformed additional studies in FC1245 cocultured with CAFs orcultured with CM of CAFs that received an extra in vitro 5 Gy. Weobserved a 1.8-fold � 0.2 increase in iNOS protein levels in FC1245

    Figure 1.

    Radiotherapy (RT) increases iNOS expression in PDAC. A, Quantification of immunofluorescence staining of iNOS in PDAC tumors with or without radiotherapytreatment (mean � SEM, n ¼ 3). Confocal images of iNOS staining and patient clinical history are described in Supplementary Fig. S1 and Supplementary Table S1.� , P < 0.05; �� , P < 0.01, compared with corresponding nonradiotherapy tissue; &, P < 0.05, compared with corresponding nontumor tissue and based on a Student ttest.B andC, Immunofluorescence staining of iNOS, cytokeratin, andaSMA in PDAC samples obtained frompatient #3 after radiotherapy. DAPIwas used to stain cellnuclei. Quantifications of iNOS/aSMAand iNOS/cytokeratin stainingwere performedusing theHalo software anddata are shown inSupplementary Tables S2 andS3.Scale bars, 50 mm. D and E, Immunofluorescence staining of iNOS, cytokeratin, and aSMA in FC1245 orthotopic PDAC tumors. DAPI was used to stain cell nuclei.Quantifications of iNOS/aSMA and iNOS/cytokeratin staining were performed using the Halo software and data are shown in Supplementary Tables S4–S7. Tumorswere collected at 2 days after a 12 Gy radiotherapy dose. Scale bars, 2,000 mm. (D) and 50 mm (E). H&E, hematoxylin and eosin.

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  • cells cocultured with CAFs or a 3.6-fold� 0.5 increase in FC1245 cellscultured with CM of CAFs (Fig. 2C). FC1245, FC1245 cultured withCM of RT-CAFs, and FC1245 cocultured with RT-CAFs demonstrat-ed, respectively, a cell viability of 92.3% � 9.42, 113.9% � 6.07, and64.94% � 12.52 (Supplementary Fig. S6).

    We next measured nitrite (NO2�), a stable byproduct of NO, in the

    culture medium of CAFs, FC1245 cells, or FC1245 cells coculturedwith CAFs (Fig. 2D). Levels of NO2

    � were higher in CAFs relative toFC1245 cells in both nonirradiated and irradiated experimental con-ditions (Fig. 2D). These results are consistent with previous studiesreporting low amounts ofNO inT3M4 andPT45-P1 pancreatic cancercells (29). Cultures or FC1245 cells with CM of RT-CAFs displayedhigher NO2

    � levels when compared with non-RT CAFs (Fig. 2D).NO2

    � secretion by FC1245 cells was strongly upregulated by CM ofradiotherapy-activated CAFs receiving an extra in vitro radiotherapy

    dose of 5 Gy (44.7 � 0.1, mean � SEM, n ¼ 4; Fig. 2D). Additionalstudies in SUIT-2, CAPAN-2, MIA PaCa-2, and BxPC3 humanpancreatic cancer cell lines cultured with CM of radiotherapy-activated human CAFs demonstrated that an in vitro radiotherapydose of 5 Gy increases NO2

    � secretion by cancer cells (SupplementaryFig. S7). These results show that radiotherapy-activated CAFs producehigh levels of iNOS and NO secretion, which are further induced byPDAC cells and that radiotherapy enhances iNOS/NO paracrinesignaling in PDACs.

    Given that inflammatory cytokines such as IFNg , IL1b, and TNFainduce iNOS expression through NF-kB (22, 29, 30), we performedanalyses of NF-kB and 32 cytokines using a MILLIPLEX kit. NF-kBactivity in FC1245 cells was monitored using a NF-kB luciferasereporter assay. Culture of FC1245 with CM from RT-CAFs increasedNF-kB in the tumor cells (Fig. 2E). Cytokine analyses were

    Figure 2.

    Radiotherapy (RT)-activated CAFs enhance PDAC tumor growth.A, CAFs were isolated from nonradiotherapy-treated FC1245 orthotopic PDACs (non-RT CAFs) orfrommice treated with a 12 Gy radiotherapy dose (RT-CAFs). FC1245 were cultured with conditioned medium from CAFs or cocultured with CAFs using a transwellin vitro system. B, Western blot of iNOS in total lysates of FC1245, non-RT CAFs, and RT-CAFs. Density of Western blot bands was quantified by scanningdensitometrywith ImageJ software.C,Western blot analysis of iNOS in total lysates of FC1245. FC1245 cellswere coculturedwith CAFs (RT-CAFs or non-RTCAFs) ortreated with CM from CAFs (RT-CAFs, CM, or non-RT CAFs, CM) for 24 hours. Density of Western blot bands was quantified by scanning densitometry with ImageJsoftware. D, NO2

    � levels in supernatants of FC1245, RT-CAFs, non-RT CAFs, FC1245 treated with supernatants from CAFs, and FC1245 cocultured with CAFs. � , P <0.05; �� , P < 0.01; ��� , P < 0.001 comparedwith FC1245 and based on a Student t test (mean� SEM, n¼ 4). E, Luciferase NF-kB activity assay (mean� SEM, n¼ 3) inFC1245 cells cultured with CM non-RT CAFs, CM RT-CAFs, and CM RT-CAFs in the presence of 10 mmol/L 1400W. �� , P < 0.01, compared with FC1245; &&, P < 0.01compared with FC1245 treatedwith CM from non-RT CAFs; $$, P < 0.01, comparedwith FC1245 treated with CM from RT-CAFs and based on a Student t test. 1400W,N-(3-(aminomethyl)benzyl)acetamidine. F, IFNg , IL1b, and TNFa concentrations in the supernatants of FC1245 PDAC cells (first bar on the graph), non-RT CAFs(second bar), RT-CAFs (third bar), FC1245 incubated with CM from non-RT CAFs (fourth bar), and FC1245 incubated with CM from RT-CAFs (fifth bar). Cellsupernatantswere collected at 24hours after cell culture and cytokine activitywasdeterminedusing theMILLIPLEXMAPMouseCytokine/ChemokineMagnetic Bead32 Plex Panel. Data are presented as mean� SEM; n¼ 4–6 independent experiments. � , P < 0.05; �� , P < 0.01, compared with FC1245 and based on a Student t test.G, Bioluminescence images and tumor growth rate (mean� SD, n¼ 4) of pancreatic orthotopic tumors developed by implantation of FC1245lucþ alone, FC1245lucþmixedwith non-RTCAFs, and FC1245lucþmixedwith RT-CAFs into the pancreas of C57BL/6 (B6)mice. � , P

  • performed using the cell supernatant of FC1245, non-RT CAFs, RT-CAFs, and FC1245 cells cultured either with medium from non-radiotherapy fibroblasts or with medium from radiotherapy fibro-blasts. IFNg , IL1b, leukemia-inhibitory factor (LIF), RANTES (reg-ulated on activation normal T-cell expressed and secreted), and IL13were similar in FC1245 and CAFs (Fig. 2F). Culture of FC1245PDAC cells in the presence of CM from radiotherapy fibroblastsincreased the production of IFNg , IL1b, and TNFa (Fig. 2F). Inaddition, when FC1245 cells were cultured with medium fromradiotherapy fibroblasts, we observed an increase in the productionof IL6, LIF, and RANTES (Supplementary Fig. S8). On the otherhand, IL13 decreases when FC1245 cells were cultured with mediumfrom nonradiotherapy fibroblasts (Supplementary Fig. S8). We nextused real-time PCR to understand the major genes in radiotherapy-activated CAFs driving these transcriptional differences in FC1245cancer cells (Supplementary Fig. S9). We observed upregulation inIFNb, iNOS, and TNFa in RT-CAFs when compared with non-RTCAFs. When compared with RT-CAFs, nonradiotherapy CAFsdemonstrated higher mRNA levels for RANTES, C-X-C motifchemokine 10 (CXCL10), IL12, IL1a, IL1b, IL1R1, IL6Ra, Inter-feron regulatory factor 7 (IRF7), and STAT1 (SupplementaryFig. S9). These results suggest that radiotherapy-activated fibroblastsinduce increased secretion of NO and iNOS expression in the tumorcells through NF-kB, which in turn leads to an elevated release ofinflammatory cytokines by the tumor cells.

    RT-CAFs increase pancreatic tumor growth, an effect that canbe abolished by genetic or pharmacologic blockade of iNOS

    Recent studies have demonstrated that PDAC tumor growth inorthotopicmurinemodels is faster in tumors developed by inoculationof tumor cells plus CAFs when compared with tumor cells alone (9). Inour study, FC1245 pancreatic orthotopic tumor models were used todetermine the role of non-RT CAFs and RT-CAFs in PDAC tumorgrowth in vivo. FC1245lucþ alone, FC1245lucþ mixed with non-RTCAFs, and FC1245lucþmixed with RT-CAFs were implanted into thepancreas of C57BL/6 (B6)mice and tumor growth was evaluated usingbioluminescence. As shown in Fig. 2G, the tumor growth rate wassignificantly higher in FC1245 cells mixed with RT-CAFs than that inthe model developed by mixing FC1245 cells with non-RT CAFs orFC1245 cells alone (Fig. 2G). At 14 days after cells' implantation in thepancreas, we observed significantly higher aSMAþ in tumors devel-oped by FC1245lucþ mixed with CAFs when compared withFC1245lucþ cells alone (Supplementary Fig. S10). To explain theprotumor effect of RT-CAFs, the influence of these cells on the growthof FC1245 cells was investigated in vitro using the Trypan blue assay.Enhanced FC1245 cell growth was observed after cells being culturedwith CM of RT-CAFs (Fig. 2H). Culture of FC1245 with CM ofnonradiotherapyCAFs did not interferewith PDACcancer cell growth(Fig. 2H). Next, we used the Boyden chamber (Fig. 2I) and scratchassays (Supplementary Fig. S11) to determine whether RT-CAFsaffected the migratory properties of FC1245 cells. Matrigel invasionchamber assays were also used to measure FC1245 invasion propertiesin the presence of CAFs (Fig. 2J). FC1245 cells in culture with CM ofRT-CAFs demonstrated a significant increase in both migration andinvasion properties as compared with FC1245 cells alone, which isconsistent with previous studies (7). Culture of FC1245 cells with CMof RT-CAFs showed a significantly larger number of migratory andinvading cells compared with culture with CM of nonirradiatedfibroblasts (Fig. 2I and J; Supplementary Fig. S11). We did not detectmetastatic spread at 6 days after cells' implantation in the pancreas(Supplementary Fig. S12). At 14 days, metastatic spread of tumor was

    observed in the liver, spleen, and stomach in tumors developed byFC1245lucþ mixed with RT-CAFs (Supplementary Fig. S13).

    Premised on our findings (Figs. 1 and 2), we expected that theprotumor effects of RT-CAFs are mediated by iNOS/NO signaling.Therefore, we sought to investigate FC1245 cells growth after iNOSamplification or exposure to physiologic levels of the NO donordiethylenetriamine NONOate (DETA-NO). DETA-NO has a half-life of 20 hours and in aqueous solution it spontaneously decomposesto produce a long lasting NO release. Previous studies have reportedthat DETA-NO increases migration of lung carcinoma cells andproliferation of breast cancer cells (26, 42). Others have reported thatlow concentrations of DETA-NO (20–2000 nmol/L) induce cancer cellgrowth, while concentrations higher than 20 mmol/L inhibit cellproliferation (34). In our studies, NO induction in PDAC cells wasperformed using 1 mmol/L of DETA-NO or with a clustered regularlyinterspaced short palindromic repeats (CRISPR) activation plasmid(Supplementary Figs. S14 and S15). Treatment of FC1245 pancreaticcancer cells with 1 mmol/L of DETA-NO or with CRISPR activationplasmid increased NO levels and FC1245 cell growth, an effect that isabolished by the addition of the highly selective iNOS inhibitor 1400W(Supplementary Figs. S14 and S15; ref. 43). To support the hypothesisthat NO release from CAFs contributes to radiotherapy resistance ofpancreatic carcinoma cells, NO induction in PDAC cells in thepresence of RT-CAFs was blocked using siRNA-mediated knockdownof iNOS or pharmacologic treatments with 1400W (Fig. 2H; Supple-mentary Figs. S14–S16). In addition, the iNOS inhibitor 1400WdecreasedNF-kB induction in tumor cells cultured with CMRT-CAFs(Fig. 2E). These results indicate that CAFs can promote the growth ofPDAC cells, an effect that increases when cells are cultured with CMofRT-CAFs, which can be abolished in vitro by iNOS depletion usinggenetic and pharmacologic approaches.

    Pharmacologic blockade of iNOS improves therapeuticresponse of PDACs to radiotherapy

    Previous studies demonstrated a role for iNOS in PDAC chemore-sistance (20). Our findings suggest iNOS/NO signaling as a cause ofPDAC resistance to radiotherapy, with NO secretion from RT-CAFspreventing radiotherapy therapeutic efficacy. To translate our findingsinto a clinically relevant approach, we performed therapeutic studiescombining iNOS pharmacologic inhibition with radiotherapy inorthotopic PDAC models. Luciferase-expressing FC1245 pancreaticcancer cells were implanted into the pancreas of C57BL/6 (B6) mice.To determine the impact of radiotherapy dose on PDAC therapy, apilot therapeutic study was performed using 12 Gy in a singletreatment fraction or 18 Gy administered in three fractions of 6 Gyat 48-hour intervals. These dose schedules were selected because, as perthe linear quadratic model of cell kill, they have similar biologicallyeffective dose (BED at an alpha/beta¼ 10, BED10). The three-fractioncourse of radiotherapy (BED10 ¼ 28.8 Gy) improved survival whencompared with a single fraction (BED10 ¼ 26.4 Gy; SupplementaryFig. S17), supporting previous reports that are suggestive of the clinicalbenefit of radiotherapy administration in fractions (11). In furthertherapeutic experiments, radiotherapywas administered in 3 doses of 6Gy at 48-hour intervals. After an engraftment period, tumor-bearingmice as identified via bioluminescence were randomly assigned totreatment groups (nonradiotherapy, radiotherapy, 1400W, and1400W/radiotherapy). At 14 days after the first radiotherapy dose,mice treated with 1400W or 1400W/radiotherapy demonstratedsignificantly decreased tumor growth and tumors of small size asmeasured by luminescence (Fig. 3A–C). The weight of tumors at14 days after the first radiotherapy dose was 1.65 � 0.60, 0.92 � 0.04,

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  • and 0.60 � 0.09 g in mice treated with radiotherapy, 1400W, and1400W/radiotherapy (Supplementary Fig. S18).

    One of the downstream effects of NO in cancer includes evasion toapoptosis (24, 44) and therefore, tumor sections of the differenttherapeutic groups were immunostained to assess proliferation andapoptosis. The 1400W/radiotherapy-treated mice displayed dimin-ished proliferation of tumor cells based on the reduced number ofKi67þ tumor cells compared with control, 1400W, and radiotherapygroups (Fig. 3D). IHC staining of cleaved caspase-3 indicated signif-icantly increased apoptosis in pancreatic tumors treated with radio-therapy and 1400W/radiotherapy (Fig. 3D). Collectively, these resultsdemonstrate that radiotherapy/iNOS inhibition slows pancreatictumor growth.

    NO secretion fromRT-CAFs changes PDAC TMEextracellular pHOne of the mechanisms by which NO modulates tumor cell

    proliferation is through upregulation of glycolysis (34). NO releaseinto tumor cells reduces mitochondrial oxidative phosphorylation atcomplexes III and IV of the electron transport chain and increasesactivity of the 6-phosphofructokinase 1 (PFK-1), which results in theactivation of glycolysis promoting a metabolic switch from an aerobic

    to a glycolytic phenotype (Warburg effect; refs. 34, 45–50). Weattempted to study changes in pancreatic cancer cells glycolysis afterFC1245 cells incubation with the NO-donor DETA-NO or culturewithCMofRT-CAFs.Our results show thatNOproduction in FC1245cells is increased through exposure to DETA-NO (SupplementaryFig. S14). Glucose consumption and lactate production (Supplemen-tary Fig. S19) are increased compared with without DETA-NOaddition. This effect was further inhibited by the addition of the iNOSinhibitor 1400W (Supplementary Fig. S19).

    Because our results showed that DETA-NO increased the glycolyticrate when compared with untreated FC1245 cells, we hypothesizedthat NO release from CAFs could increase glucose consumption andlactate secretion in cancer cells. Cultures of FC1245 with CM of CAFsdemonstrated that RT-CAFs led to a significant increase in glucoseuptake (1.3� 0.1, mean� SEM, n¼ 4; Fig. 4A) and lactate production(2.1� 0.6, mean� SEM, n¼ 4; Fig. 4A). At 14 days postradiotherapy,we observed an increase in GLUT1 expression of PDAC tumors (1.7�0.2, mean � SEM, n ¼ 3; Fig. 4B). Additional immunofluorescencestaining of aSMA, cytokeratin, and GLUT1 in radiotherapy-treatedPDAC samples demonstrated GLUT1þ/aSMAþ in 8.4% of totalaSMAþ cells and GLUT1þ/cytokeratinþ in 34.3% of total

    Figure 3.

    Pharmacologic blockade of iNOS improves therapeutic response of PDACs to radiotherapy. A-C, FC1245 luciferase–expressing PDAC orthotopic tumorstreated with radiotherapy, 1400W, or 1400W/radiotherapy (n¼ 10 mice per group) and tracked by bioluminescence. Mice were treated with 3 doses of 6 Gy at48-hour intervals. Intraperitoneal administration of 1400W (1 mg/g) was started at day 0. 1400W was administered every day for 7 days during theradiotherapy treatment period and every two days during two weeks after the radiotherapy treatment period. Real-time images from median of two animals(B) and luminescent signal (n¼ 10 mice per group) on day 14 posttherapy (C). �� , P < 0.01, compared with control and based on a Student t test (A). �� , P < 0.01;��� , P < 0.01, compared with radiotherapy-treated group and based on a Student t test. &, P < 0.01, compared with 1400W-treated group (C).D, Hematoxylin and eosin (H&E), cleaved caspase-3, and Ki-67 IHC and quantification (mean � SEM, n ¼ 3) in tumors from control, radiotherapy,1400W, and 1400W/radiotherapy groups collected on day 14 posttherapy. � , P < 0.05; �� , P < 0.01, compared with control; &, P < 0.05, compared withradiotherapy; $, P < 0.05; $$, P < 0.01, compared with 1400W and based on a Student t test.

    iNOS Regulates PDAC Response to Radiotherapy

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  • cytokeratinþ cells (Fig. 4C andD; Supplementary Tables S8–S11). Thepercentage of GLUT1þ/cytokeratinþ cells was 7.6-fold higher inradiotherapy-treated samples when compared with control (Fig. 4Cand D; Supplementary Tables S8–S11). We next used fluorodeoxy-glucose (18F-FDG) positron emission tomography (PET) to validatein vivo changes in GLUT1 and glucose consumption observedin Fig. 4A–D. Our biodistribution and PET imaging results at 14 dayspost first radiotherapy dose in mice bearing FC1245 orthotopic PDAC

    tumors demonstrated a significant increase in 18F-FDGPDAC uptake,an effect that decreases in mice treated with radiotherapy/1400W(Fig. 4E and F; Supplementary Fig. S20). As glycolysis promotesintracellular Hþ generation contributing to the membrane dynamicsof Naþ/Hþ flux, as mediated by the sodium/hydrogen exchangerisoform 1 (NHE1), an increase in glycolysis as mediated by NO releasefrom RT-CAFs could activate NHE1. Our results revealed that radio-therapy increases NHE1 expression in FC1245 PDACs, an effect that

    Figure 4.

    Radiotherapy-activated CAFs change PDAC-TME extracellular pH, allowing detection with pH-targeted pHLIP. A, Glucose uptake and lactate production of FC1245PDAC cells treated with CM from CAFs. Data are presented as mean� SEM, n¼ 4. � , P < 0.05, compared with FC1245 and based on a Student t test. NBDG, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose. B–D, Western blot analysis of GLUT1 (B) and immunofluorescence of GLUT1/aSMA (C) and GLUT1/cytokeratin (D) in total lysates of FC1245 orthotopic PDAC tumors obtained at 14 days postradiotherapy. Density ofWestern blot bands was quantified by scanningdensitometry with ImageJ software. E and F, PDAC tumor uptake (E) and maximum intensity projection PET images (F) at 2 hours after injection of 18F-FDG in micebearing FC1245 orthotopic tumors. At 14 days posttherapy, mice were administered 200 mCi intravenous injection of 18F-FDG. Bars, n ¼ 4 mice per group; mean �SEM. %ID, percentage of injected dose. G, Confocal images and quantification (mean � SEM, n ¼ 3) of immunofluorescence staining of NHE1 (green) in FC1245orthotopic PDAC tumors obtained at 14 days postradiotherapy. DAPI (blue) was used to stain cell nuclei. Scale bars, 50 mm. H and I, PDAC tumor uptake asdetermined by acute biodistribution studies at 24 hours after injection of 67Ga-labeled pHLIP inmice bearing FC1245 orthotopic tumors. At 14 days postradiotherapy,micewere intravenously given 20 mCi of 67Ga-labeled pHLIP. Bars, n¼ 4mice per group;mean� SEM. %ID/g, percentage of injected dose per gram. J,An increase inglycolysis as mediated by NO release from RT-CAFs activates NHE1. Activation of NHE1 causes a reversal of the plasma membrane pH gradient, resulting in a morealkaline intracellular pH and a more acidic extracellular pH that can be detected using pH-targeted molecular imaging with the pH (low) insertion peptide (pHLIP)technology.

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  • was inhibited with the iNOS inhibitor 1400W (Fig. 4G). In sum, ourfindings indicate that an increase in iNOS/NO signaling in RT-fibroblasts upregulates PDAC glycolysis.

    pH-targeted pHLIP allows assessment of radiotherapy/1400Wresponse within PDAC

    Activation of NHE1 causes a reversal of the plasma membranepH gradient (51), resulting in a more alkaline intracellular pH anda more acidic extracellular pH (Fig. 4H–J). As we observed anincrease in GLUT1 and NHE1 in radiotherapy-treated tumors(Fig. 4A–G), we attempted to use a pH-targeted imaging probe todetect changes in the extracellular microenvironment pH, as medi-ated by NO secretion from RT-CAFs. pH (Low) Insertion Peptide(pHLIP) allows detection of tumor cell surface acidity (Fig. 4H;ref. 52). We performed in vivo biodistribution studies with theradiometallated pHLIP variant [67Ga]Ga-NO2A-cysVar3 in micebearing FC1245 orthotopic PDAC tumors. On the basis of ourstudies demonstrating that at 14 days posttherapy, the tumor growthwas slowed in mice treated with 1400W/radiotherapy when com-pared with radiotherapy alone (Fig. 3A–C), mice were administeredwith [67Ga]Ga-NO2A-cysVar3 at 14 days post first radiotherapydose. At 24 hours postinjection of [67Ga]Ga-NO2A-cysVar3, PDACradiotherapy-treated tumors had an uptake of 8.6 � 0.7 percentageinjected dose per gram tissue (%ID/g, mean � SEM, n ¼ 5),while control tumors had an uptake of 3.6% � 1.9% ID/g,mean � SEM, n ¼ 5 (Fig. 4I; Supplementary Fig. S21). Theincrease in [67Ga]Ga-NO2A-cysVar3 PDAC tumor uptake, due toan increase in extracellular acidity, was further decreased in micetreated with radiotherapy plus 1400W (3.1 ID/g� 1.0, mean� SEM,n ¼ 5, Fig. 4I). Taken together, these results suggest that NOsignaling activation in radiotherapy-treated tumors will result inchanges of the PDAC extracellular pH (Fig. 4J) that can be detectedusing radiolabeled pHLIP, suggesting a clinical potential of pHLIPto assess radiotherapy response within pancreatic cancer, and sug-gesting ultimately that the benefit of radiotherapy intervention inPDAC can be improved by blocking NO signaling.

    DiscussionThemechanisms of iNOS/NO signaling and their actions on PDAC

    response to radiotherapy remain to be completely understood. Here,we demonstrate that radiotherapy enhances iNOS expression andsubsequent NO secretion in PDAC.Mechanistic studies demonstratedthat the CM of irradiated CAFs drives the production of iNOS inPDACs. Cultures of PDAC cells with CM of RT-CAFs demonstratethat NO secretion and iNOS expression by RT-CAFs are furtherinduced by pancreatic tumor cells through NF-kB, which in turnleads to the release of inflammatory cytokines by the tumor cells. Inaddition, we found that NO increases after radiotherapy in PDAC,results in acidification of themicroenvironment, which can be detectedby molecular imaging using pHLIP. These data support that NO has aprotumor function in irradiated PDACs by promoting a glycolyticphenotype as detected in vivo by 18F-FDGPET imaging, which leads toextracellular acidosis. iNOS pharmacologic inhibition or knockdownimproved radiotherapy efficacy and decreased PDAC tumor growth,suggesting an integral role for NO and radiotherapy-mediated iNOSactivity in PDAC therapy. Until our study, a role for radiotherapy-mediated iNOS/NO signaling in PDAC remained unexplored.

    NOS exists as three isoforms: inducible NOS (iNOS or NOS2),neuronal NOS (nNOS or NOS1), and endothelial NOS (eNOS orNOS3). nNOS and eNOS produce low amounts of NO and are

    constitutively expressed by neuronal and vascular endothelial cells.iNOS produce a higher and sustained level of NO and are transcrip-tionally regulated and induced by inflammatory cytokines (21, 22, 29).The expression of iNOS and production of NO have been detected inseveral tumor types and are comprehensively reviewed elsewhere (22).Our findings show that, in contrast to PDAC samples, nontumortissues exhibit lower iNOS expression levels (Figs. 1 and 2; refs. 20, 28).Previous studies have demonstrated that iNOS expression is found notonly in the tumor, but also in CAFs, inflammatory, and endothelialcells (29). Further studies are necessary to evaluate the consequences ofradiotherapy on NOS expression in other noncancer cells that con-tribute to the stromal compartment of the PDAC TME.

    While some studies have demonstrated that tumor cell–derived NOinhibits tumor progression (22, 23, 26, 42, 53, 54), others havedemonstrated its role in the development of tumor growth (22, 55, 56).Similar to what has been observed for other tumor types, the role NOplays in PDAC is also not clearly defined. PDAC tumor cells contain-ing low levels of NO exhibit an aggressive metastatic phenotype whencompared with tumor cells containing high NO levels (21) andtreatment with a NO-donor inhibited proliferation and invasion ofPDAC (54). Other studies have reported an association between highiNOS expression and poorer survival in patients with PDAC (20) and arole for iNOS/NO signaling in PDAC chemoresistance (29). Con-versely, low iNOS levels are associated with enhanced survival in KPCmice containing orthotopic PDAC (20). Our studies clearly establishthat radiotherapy increases iNOS expression and NO signaling inPDAC contributing to tumor resistance to therapy.

    The contribution ofNO to tumor progression depends on its source:tumor cells, tumor-associated stromal fibroblasts, and immune cells.Radiotherapy-mediated cellular damage of surviving CAFs (6, 16)alters the paracrine interactions between tumor and stroma throughalterations in inflammatory secretome (6). To determine the cellularsources responsible for iNOS/NO increase in PDAC after radiother-apy, we performed in vitro studies using pancreatic tumor cells inculture with CAFs in a transwell coculture system or with CM of thesefibroblasts. Our data show that, although undetectable in radiother-apy-activated tumor cells, RT-CAFs showed significant iNOS expres-sion and NO secretion (Fig. 2B–D). In our experiments, an extrain vitro radiotherapy dose was necessary to study the mechanisms bywhichRT-CAFs induce iNOS/NOsecretion in the tumors cells (Fig. 2).When compared with non-RT-CAFs, RT-CAFs demonstrated upre-gulation in IFNb, iNOS, and TNFa (Supplementary Fig. S9). Cocul-ture of PDAC cells with RT-CAFs and culture with CM of CAFsincreased iNOS expression/NO secretion in the tumors cells (Fig. 2Cand D). The increase in iNOS expression and NO secretion by thetumor cells was higher when cells were cultured with CAF-CM whencompared with cells cocultured with CAFs (Fig. 2C and D). Theseresults suggest that the secretome of RT-CAFs is the major drivingforce in the production of iNOS in PDACs. The differences in iNOS/NO between FC1245 cocultures and cultures with CAF-CM can beexplained by changes in PDAC cells viability (Supplementary Fig. S6)and future studies are necessary to determine the temporal dynamics ofiNOS/NO signaling in these in vitro culture systems.

    RT-CAFs increased PDAC cell growth, migration, invasion, andmetastatic spread of tumor (Fig. 2G–J; Supplementary Fig. S13).Blocking of NO release, by iNOS inhibition with 1400W or usingknockdown via siRNA, abolished the protumor effect of RT-CAFs.Admittedly, while we successfully demonstrate that CAFs play a role inthe radiotherapy-mediated increase of iNOS/NO signaling in PDACin vitro, this system does not fully reflect the complexity and cellulardiversity of the PDAC microenvironment in vivo.

    iNOS Regulates PDAC Response to Radiotherapy

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  • Because NO regulates several biochemical pathways, the molec-ular mechanisms by which NO increases in CAFs after radiother-apy, and how iNOS/NO signaling can be further induced by thetumor cells facilitating tumor growth (Fig. 2E) are likely broad. Atthe transcriptional level, TNFa, IFNg , IL6, and IL1b are all knownto activate NF-kB, which induces iNOS expression (22, 29, 30).Here, we show that the protumor inducing effect of radiotherapy-activated fibroblasts occurs through NF-kB and involves increasedsecretion of IFNg , IL1b, TNFa, IL6, and LIF (an IL6 class cytokine)by pancreatic tumor cells. In addition, our studies demonstrate thatthe iNOS inhibitor 1400W decreases NF-kB induction in PDACscultured with secretome from RT-CAFs.

    Preclinical and clinical studies have shown low toxicity of iNOSinhibitors (33, 57–59), suggesting their potential as therapeutic agents.For instance, iNOS inhibitors have been show to block colon adeno-carcinoma (60) and glioma tumor stem cell tumor growth (33). Ourfindings that radiotherapy increases iNOS expression, suggests iNOSinhibition as a promising strategy that could be applied in thetreatment of PDAC. In our studies, the iNOS inhibitor 1400W delayedtumor growth, an effect that was enhanced with the 1400W/radio-therapy combination therapy (Fig. 3A–C). Importantly, the 1400W/radiotherapy therapeutic approach shows potential to increase pres-ervation of healthy tissues during surgical PDAC interventions due tothe observation of increasingly localized tumors with less diffusemargins (Fig. 3B). The 1400W/radiotherapy combination therapywas unable to completely eliminate tumor growth and further studiesare necessary to determine whether other iNOS inhibitors or combi-nation of iNOS inhibition with other treatment regimens may furtheraugment efficacy when compared with 1400W/radiotherapy. Previousreports in orthotopic intracranial tumors demonstrated highertherapeutic efficacy for the iNOS inhibitor BYK191023 whencompared with 1400W (33) plausibly due to its improvedpharmacokinetic and bioavailability properties.

    In ovarian cancer cells, NO decreases mitochondrial respiration,which results in the activation of glycolysis promoting a metabolicswitch from an aerobic to a glycolytic phenotype (34). Glycolysispromotes Hþ generation, which is removed from the intracellular tothe extracellular compartments in exchange with Naþ, by NHE1 (51).In our studies, we observed thatNO increases glycolytic rate andNHE1expression in PDAC (Fig. 4A–G). These membrane transportmechanisms contribute to the acidification of the extracellular space.Equipped with this information, we used a radiolabeled peptide,[67Ga]Ga-NO2A-cysVar3 pHLIP to probe changes in the TME extra-cellular pHarising from radiotherapy-mediatedNO secretion (Fig. 4Hand I). The var3 pHLIP was used in our studies due to its remarkableability to target acidity in vivo (52, 61–63) and a radiolabeled version ofvar3 pHLIP will be entering in the clinic in the current year of 2020.The radiolabeled var3 pHLIP demonstrated higher targeting accumu-lation in radiotherapy PDAC tumors when compared with unirradi-ated PDAC tumors (Fig. 4I). This increase in acidity, due to NOsecretion from radiotherapy-activated PDACs, was further decreasedwhen radiotherapy is combined with the iNOS inhibitor 1400W.

    In conclusion, our data support the notion that the secretome ofRT-CAFs initiates a paracrine activation loop increasing iNOS/NOsignaling in PDAC and additional in vitro radiation is necessary for

    robust iNOS expression by the tumor cells. Our preclinical resultsdemonstrate the potential of iNOS inhibition with fractionated radio-therapy regimen for improved response, and additionally that thatresponse can be assessed via radiolabeled pHLIP, highlighting themechanisms by whichNO in the secretome of RT-CAFs contributes toPDAC resistance. Our study supports previous reports demonstratingthat the secretome of therapy-activated CAFs contribute to PDACpathology and that targeting iNOS/NO signaling may be a validtherapeutic approach for improving radiotherapy outcomes in PDAC.

    Disclosure of Potential Conflicts of InterestT. Merghoub is a consultant at Pfizer, reports receiving a commercial research

    grant from IMVAQ, Bristol-Myers Squibb, Surface Oncology, Kyn Therapeutics,Infinity Pharmaceuticals, Peregrine Pharmeceuticals, Adaptive Biotechnologies, LeapTherapeutics, and Aprea and has ownership interest (including patents) in oncolyticviral therapy, alpha virus–based vaccine, neoantigen modeling, CD40, GITR, OX40,PD-1, and CTLA-4. J.S. Lewis has ownership interest (including patents) in pHLIP,Inc. No potential conflicts of interest were disclosed by the other authors.

    Authors’ ContributionsConception and design: P.M.R. Pereira, T. Merghoub, J.S. LewisDevelopment of methodology: P.M.R. Pereira, K.J. Edwards, F.E. EscorciaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): P.M.R. Pereira, K. Mandleywala, L.M. Carter, L.F. Campesato,M. Cornejo, L. Abma, A.-A. Mohsen, J.S. LewisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.M.R. Pereira, L.M. Carter, F.E. Escorcia,L.F. Campesato, L. Abma, T. Merghoub, J.S. LewisWriting, review, and/or revision of the manuscript: P.M.R. Pereira, K.J. Edwards,L.M. Carter, F.E. Escorcia, C.A Iacobuzio-Donahue, T. Merghoub, J.S. LewisAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): P.M.R. Pereira, J.S. LewisStudy supervision: J.S. Lewis

    AcknowledgmentsThe authors acknowledge the Radiochemistry andMolecular Imaging Probe Core,

    the Antitumor Assessment Core, and Molecular Cytology Core Facility, which weresupported by NIH grant P30 CA08748. This study was supported in part by theGeoffrey Beene Cancer Research Center of MSKCC (to J.S. Lewis) and NIH NCI R35CA232130 (to J.S. Lewis). We gratefully acknowledge Mr. William H. and Mrs. AliceGoodwin and the Commonwealth Foundation for Cancer Research and The Centerfor Experimental Therapeutics of MSKCC. P.M.R. Pereira acknowledges the TowFoundation Postdoctoral Fellowship from theMSKCCCenter forMolecular Imagingand Nanotechnology and the Alan and Sandra Gerry Metastasis and Tumor Eco-systems Center of MSKCC. L.M. Carter acknowledges support from the Ruth L.Kirschstein National Research Service Award postdoctoral fellowship (NIH F32-EB025050). T. Merghoub and L.F. Campesato are supported by Swim AcrossAmerica, Ludwig Institute for Cancer Research, Ludwig Center for Cancer Immu-notherapy at Memorial Sloan Kettering, Cancer Research Institute, Parker Institutefor Cancer Immunotherapy andBreast Cancer Research Foundation. Figures 2A, 4H,and 4J were created with Biorender. We thank Dr. Russel and Dr. Monette for theirhelp with the Arc Therapy and image analysis, respectively. We thank Egger forcutting the human samples used in this work. We thank Carl DeSelm in the MichelSadelain lab for generating the FC1245lucþ PDAC cells.

    The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

    Received September 24, 2019; revised December 30, 2019; accepted February 17,2020; published first February 21, 2020.

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  • 2020;80:1681-1692. Published OnlineFirst February 21, 2020.Cancer Res Patricia M.R. Pereira, Kimberly J. Edwards, Komal Mandleywala, et al. Cells to RadiotherapyiNOS Regulates the Therapeutic Response of Pancreatic Cancer

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