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 fibroblasts (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 expression and nitric oxide (NO) secretion were significantly 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-κB, which, in turn, elevated the release of inflammatory 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 inhibition was combined with radiotherapy. These data show the important role that iNOS/NO signaling plays in the effectiveness of radiotherapy to treat PDAC tumors.

Significance:

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.

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 nonspecific presenting symptoms, difficulty of imaging early-stage tumors, and lack of tumor biomarkers with both good specificity 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 combinations of existing therapies to expand treatment options for PDAC. Emerging therapeutic approaches for PDAC include immunotherapies, 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 combination 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 profile of tumor microenvironment (TME) that results in changes in tumor invasion, tumor growth, inflammatory mediators, and regulators of angiogenesis (6–8). Noncancer cells including immune cells, fibroblasts, pericytes, 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 inflammatory (10); radiotherapy alters the inflammatory TME by contributing to a fibroinflammatory desmoplasia (11) and to an increase in the expression of TNFα (12). The proinflammatory cytokines IFNα, IFNβ, and IFNγ are further induced in radiotherapy-treated cells (13, 14). Cancer-associated fibroblasts (CAF) expressing both vimentin and α-smooth muscle actin (αSMA) are the predominant cellular components of the PDAC stroma (2). Previous studies have demonstrated that irradiated pancreatic CAFs exhibit enhanced tumor–stroma interactions, which impact tumor growth and invasion (7, 15). Pancreatic CAFs survive severe stress, including damage induced by radiotherapy single-fraction doses up to 20 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 by inflammatory cytokines (21, 22), contributing to the production of nitric oxide (NO) through conversion of l-arginine into citrulline in the presence of NADPH and oxygen. An increase in NO results in an intrinsic prooxidant environment (22), immune escape (23), and resistance to apoptosis (24). NO shows promise either as a standalone therapeutic agent or as target of cancer therapies. The role of NO in cancer depends on the NO concentration or duration of NO exposure, extracellular conditions, localization of NOS, and cellular sensitivity to NO (21, 25–27). Pancreatic tumors exhibit higher expression of iNOS compared with nontumor pancreatic tissue (28). Pancreatic orthotopic implantation of tumor cells expressing low levels of iNOS result in the formation of pancreatic tumors with metastasis in the liver and formation of ascites—an effect that is not observed in tumor models developed by orthotopic implantation 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 fibroblasts and immune cells (22). Mechanistic studies have demonstrated that in PDAC, CAFs express high amounts of iNOS contributing to the development of tumor chemoresistance via increased NO secretion (29). Inflammatory cytokines induce NO production by CAFs (29), contributing to PDAC chemoresistance (30) and immunosuppression (31).

Not only does NO contribute to tumor growth and metastasis (21, 26, 32, 33), but mechanistic metabolic studies demonstrated a NO-mediated decrease in mitochondrial respiration, which led cancer cells to undergo higher glycolytic rates to maintain ATP production levels (34). These results have further supported the role of NO in cancer metabolism by demonstrating that NO regulates the Warburg effect and promotes cancer growth by inhibiting mitochondrial respiration.

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

Pancreatic cancer cell lines and patient samples

SUIT-2, CAPAN-2, MIA PaCa-2, and BxPC-3 human PDACs were purchased from ATCC and were grown according to standard procedures. The FC1245 murine PDACs were obtained from the David Tuvenson's group and were transformed in Michel Sadelain's laboratory to generate FC1245luc+ (35). FC1245 and FC1245luc+ cells were cultured in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin. Cell lines used in this work were purchased in 2016–2019, and they were used within passage number of 15. All the cell lines were Mycoplasma free and authenticated at Memorial Sloan-Kettering Cancer Center (MSKCC, New York, NY) integrated genomics operation core using short tandem repeat analysis.

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

Murine orthotopic PDAC xenograft model

All experiments involving animals were performed according to the guidelines approved by the Research Animal Resource Center and Institutional Animal Care and Use Committee at MSKCC. PDAC orthotopic models were developed as described previously (36) and additional details are given in Supplementary Information.

Contrast injection and CT for pancreatic tumor delineation

Pancreatic tumors were visualized using an image-guided small-animal micro-irradiator (XRad225Cx, Precision X-Ray) following a protocol previously described by Thorek and colleagues (37). Mice were injected with a 3 mL solution of 75 mg Iodine/mL (iohexol; Omnipaque, GE Healthcare) at 5 minutes prior to imaging. Data visualization was performed as described previously (37) and tumor volumes were determined using Amira region-grow tool.

Image-guided X-ray radiotherapy of pancreatic tumors

Immediately after visualization of the pancreatic tumor by X-ray CT using a solution of 15 mg iodine/mL, radiation arc therapy was performed in mice bearing orthotopic pancreatic tumors (37). See Supplementary Information for details.

Isolation and expansion of murine and human pancreatic fibroblasts

Fibroblasts were isolated from orthotopic FC1245 PDAC models or human PDAC using methodology described previously by Müerköster and colleagues (29). Further details are described in Supplementary Information.

Conditioned medium from irradiated and nonirradiated cells

To analyze the effects of radiotherapy on NO secretion from fibroblasts on tumor cells and vice versa, cells were cultured with the respective conditioned medium obtained from fibroblasts for 24 hours. Before use, these media was centrifuged at 10,000 rpm for 10 minutes.

Coculture of irradiated and nonirradiated pancreatic tumor cells with fibroblasts

FC1245 pancreatic tumor cells (1 × 105 tumor cells per well) were plated into the bottom compartment of a 6-well culture plate (Corning). The fibroblasts were then seeded into the top transwell compartment (2 × 105 fibroblasts per well). Medium was replaced after 24 hours, cocultures were irradiated (see below for details on in vitro radiotherapy treatments), and supernatants or cell extracts were analyzed after an additional 24-hour coculture. The supernatants were centrifuged at 10,000 rpm for 10 minutes before use on the analysis.

Nitrite level determination by colorimetric assays

NO is a gaseous free radical with a short life and no available methods to directly measure NO levels exist. Therefore, the levels of 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; Cayman Chemical). See Supplementary Information for details.

In vitro therapeutic treatments

Details on the different in vitro therapeutic experiments—radiation treatment, iNOS inhibition with 1400W, iNOS knockdown, iNOS amplification, NO donor treatments, glucose uptake, lactate production, cell counting, viability assessment with Trypan blue exclusion, migration, and invasion assays—are described in Supplementary Information.

NFκB, cytokine, and RNA analyses

The activity of the NFκB in FC1245 cells cultured in the presence and absence of CM from CAFs was evaluated using a NFκB reported kit 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 from fibroblasts were collected, centrifuged at 16,000 × g for 10 minutes, and saved at −80 °C until the day of the experiment. Cytokines were quantified using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead 32 Plex Panel (Millipore).

For RNA analyses, details are described in Supplementary Information. 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.

In vivo therapy studies

Bioluminescence was measured for 5 minutes after intraperitoneally administering 150 mg/kg of potassium d-luciferin salt (Caliper Life Sciences) dissolved in PBS. Mice were deemed to have engrafted tumors (typically 7 days postorthotopic implantation of FC1245 PDAC cells) once total bioluminescence surpassed 105 photons/second/cm2/sr. Mice were randomly grouped into treatment groups (n = 10 per group): vehicle, 1400W, radiotherapy, or a combination of 1400W with radiotherapy. Radiotherapy groups received three doses of 6 Gy (administered with a 48-hour interval, see sections above for details on radiotherapy protocols). Intraperitoneal administration of 1400W (1 μg/g) was started at day 0. 1400W was administered once daily for 7 days during the radiotherapy treatment period and every 2 days for 2 weeks after the radiotherapy treatment period.

Western blotting, immunofluorescence, and IHC

Western blot, immunofluorescence, and IHC were performed as detailed in Supplementary Information.

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

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

NO2A-variant 3 pHLIP (>95% chemical purity) was purchased and used as received from CSBio Co. For peptide radiolabeling with Gallium-67 (67Ga), 67Ga (gallium-67 citrate, Nuclear Diagnostic Products) 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/L Na2CO3. NO2A-cysVar3 was dissolved in DMSO and added to the [67Ga]GaCl3. Following addition of 200 μL 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). After washing the cartridge with 10 mL of water to remove unbound 67Ga, the radiolabeled peptide was eluted with 100% ethanol and reconstituted in PBS for in vivo studies. The radiochemical purity of all constructs used in animal studies exceeded 95%.

At 14 days posttherapy (see above for details on the radiotherapy protocol and 1400W administrations), mice (n = 5 per group) were administered 20 μCi intravenous injection of 67Ga-labeled pHLIP or 200 μCi 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 after injection of 67Ga-labeled pHLIP or 2 hours after intravenous injection of 18F-FDG. Mice were sacrificed and organs were harvested, weighed, and assayed in the gamma counter for biodistribution studies. Radioactivity associated with each organ was expressed as percentage of injected dose per gram of organ (%ID/g).

Radiotherapy increases iNOS expression in murine and human pancreatic tumors

Given that a higher iNOS expression is associated with poor survival in patients with PDAC (20) and with PDAC therapeutic resistance (29), we sought to determine whether radiotherapy interferes with iNOS protein levels in pancreatic cancer. We used immunofluorescence assays to characterize iNOS protein levels in matched normal versus tumor pancreatic samples from patient specimens obtained before treatment [treatment-naïve, nonradiotherapy (non-RT)] and after radiotherapy (Fig. 1A; Supplementary Fig. S1). The radiotherapy-treated PDAC samples were obtained between 1 and 3 weeks after the last radiotherapy treatment fraction (Supplementary Table S1) and the number of cumulative radiation dose given to those patients decreased in the following order patient #1 > patient #2 > patient #3 > patient #4. The tumor tissues in our four different patient cohorts showed a significantly higher iNOS expression when compared with the nontumor pancreas (Fig. 1A; Supplementary Fig. S1). These observations are consistent with previous IHC staining showing higher iNOS expression in tumor when compared with nontumor samples in PDAC cases (20, 28). The iNOS protein levels were higher in radiotherapy-treated tumors when compared with nontreated PDAC 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 iNOS was similar in nonradiotherapy versus radiotherapy-treated nontumor tissues of patients #2 and #3. Nontumor samples of 2 other patients showed a 2-fold increase in iNOS protein levels after radiotherapy (2.4 ± 0.3 patient #1 and 2.3 ± 0.5 patient #4). Additional immunofluorescence staining of αSMA and cytokeratin in radiotherapy-treated samples obtained from patient #3, demonstrated iNOS+/αSMA+ in 56.5% of total αSMA+ cells and iNOS+/cytokeratin+ in 90.1% of total cytokeratin+ cells (Fig. 1B and C; Supplementary Tables S2 and S3).

Figure 1.

Radiotherapy (RT) increases iNOS expression in PDAC. A, Quantification of immunofluorescence staining of iNOS in PDAC tumors with or without radiotherapy treatment (mean ± SEM, n = 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 t test. B and C, Immunofluorescence staining of iNOS, cytokeratin, and αSMA in PDAC samples obtained from patient #3 after radiotherapy. DAPI was used to stain cell nuclei. Quantifications of iNOS/αSMA and iNOS/cytokeratin staining were performed using the Halo software and data are shown in Supplementary Tables S2 and S3. Scale bars, 50 μm. D and E, Immunofluorescence staining of iNOS, cytokeratin, and αSMA in FC1245 orthotopic PDAC tumors. DAPI was used to stain cell nuclei. Quantifications of iNOS/αSMA and iNOS/cytokeratin staining were performed using the Halo software and data are shown in Supplementary Tables S4–S7. Tumors were collected at 2 days after a 12 Gy radiotherapy dose. Scale bars, 2,000 μm. (D) and 50 μm (E). H&E, hematoxylin and eosin.

Figure 1.

Radiotherapy (RT) increases iNOS expression in PDAC. A, Quantification of immunofluorescence staining of iNOS in PDAC tumors with or without radiotherapy treatment (mean ± SEM, n = 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 t test. B and C, Immunofluorescence staining of iNOS, cytokeratin, and αSMA in PDAC samples obtained from patient #3 after radiotherapy. DAPI was used to stain cell nuclei. Quantifications of iNOS/αSMA and iNOS/cytokeratin staining were performed using the Halo software and data are shown in Supplementary Tables S2 and S3. Scale bars, 50 μm. D and E, Immunofluorescence staining of iNOS, cytokeratin, and αSMA in FC1245 orthotopic PDAC tumors. DAPI was used to stain cell nuclei. Quantifications of iNOS/αSMA and iNOS/cytokeratin staining were performed using the Halo software and data are shown in Supplementary Tables S4–S7. Tumors were collected at 2 days after a 12 Gy radiotherapy dose. Scale bars, 2,000 μm. (D) and 50 μm (E). H&E, hematoxylin and eosin.

Close modal

To further compare iNOS protein levels between nonradiotherapy and radiotherapy-treated pancreatic tumors, FC1245luc+ murine pancreatic cancer cells were orthotopically transplanted to the pancreas of C57BL/6 (B6) mice. FC1245 pancreatic cancer cells isolated from KPC (KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre) mice in the C57BL/6 (B6) genetic background represent a valuable biological PDAC model (38) as they mimic the pathophysiologic features of human PDAC, and when transplanted into mice they develop invasive metastatic PDAC characterized by extensive stroma (39). Murine radiotherapy treatment planning utilized CT imaging with intraperitoneally administered contrast agent to define the orthotopic PDAC tumor area and the entire tumor mass was selectively irradiated with a single radiotherapy therapeutic absorbed dose of 12 Gy using an arc treatment (37). The use of a 12 Gy dose to study radiotherapy-mediated iNOS production was based on previous studies demonstrating that radiotherapy at this dose accelerates the progression of PDAC invasion (11), which we hypothesize will correspondingly effectuate radiotherapy-mediated iNOS production. Western blot (Supplementary Fig. S2) and immunofluorescence (Fig. 1D) analyses of murine orthotopic PDACs obtained at 2 days after radiotherapy confirms the increase in iNOS protein levels (13.2-fold ± 0.1, mean ± SEM, n = 4). Radiotherapy-treated PDAC exhibited a 5-fold increase in SMA when compared with non-radiotherapy PDAC (Fig. 1D; Supplementary Tables S4 and S5). Quantification of iNOS colocalization with αSMA and cytokeratin in FC1245 radiotherapy-treated tumors demonstrated a 2-fold increase iNOS+/αSMA+ and a 5-fold increase iNOS+/cytokeratin+ when compared with non-radiotherapy PDAC (Fig. 1D and E; Supplementary Tables S4–S7). Additional IHC studies 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 12 Gy exhibit fibroinflammatory desmoplasia associated with an increase in collagen and αSMA (11), suggesting pancreatic stellate cell activation. Together, these results suggest that local tumor irradiation induces iNOS expression in pancreatic tumors.

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

Pancreatic TME largely consists of stromal fibroblasts, which are thought to contribute to PDAC resistance to treatment (40, 41). Therefore, we examined the role of CAFs in iNOS expression and NO secretion of PDAC following radiotherapy. Previous studies have demonstrated increased invasiveness of pancreatic cancer cells after coculture with fibroblasts irradiated at a dose of 5 Gy (7). We performed primary cultures of fibroblasts (29) isolated from surgical resections of treatment naïve (nonradiotherapy) and radiotherapy-treated FC1245 PDACs (Supplementary Fig. S4A and S4B). To investigate possible paracrine iNOS/NO signaling, we analyzed CM of radiotherapy versus nonradiotherapy CAFs and in vitro coculture models consisting of FC1245 pancreatic cancer cells and CAFs (Fig. 2A; Supplementary Fig. S5). To mimic the high PDAC desmoplastic stromal environment, we followed previous reports and used a tumor cell–CAF ratio of 1:2 in our in vitro model system (29). Western blot analysis demonstrated similar iNOS expression in FC1245 cancer cells and CAFs (Fig. 2B). Additional studies in FC1245 cancer cells and CAFs that received an extra in vitro irradiation dose of 5 Gy demonstrated, respectively, a 2.2-fold and 3.3-fold increase in iNOS protein levels in non-RT CAFs and RT-CAFs (Fig. 2B). iNOS expression was similar 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 incubated with CM of CAFs for 24 hours (Fig. 2C). Because an extra in vitro irradiation dose increases iNOS expression in CAFs (Fig. 2B), we performed additional studies in FC1245 cocultured with CAFs or cultured with CM of CAFs that received an extra in vitro 5 Gy. We observed a 1.8-fold ± 0.2 increase in iNOS protein levels in FC1245 cells cocultured with CAFs or a 3.6-fold ± 0.5 increase in FC1245 cells cultured with CM of CAFs (Fig. 2C). FC1245, FC1245 cultured with CM of RT-CAFs, and FC1245 cocultured with RT-CAFs demonstrated, respectively, a cell viability of 92.3% ± 9.42, 113.9% ± 6.07, and 64.94% ± 12.52 (Supplementary Fig. S6).

Figure 2.

Radiotherapy (RT)-activated CAFs enhance PDAC tumor growth. A, CAFs were isolated from nonradiotherapy-treated FC1245 orthotopic PDACs (non-RT CAFs) or from mice treated with a 12 Gy radiotherapy dose (RT-CAFs). FC1245 were cultured with conditioned medium from CAFs or cocultured with CAFs using a transwell in 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 scanning densitometry with ImageJ software. C, Western blot analysis of iNOS in total lysates of FC1245. FC1245 cells were cocultured with CAFs (RT-CAFs or non-RT CAFs) or treated 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 ImageJ software. 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 compared with FC1245 and based on a Student t test (mean ± SEM, n = 4). E, Luciferase NF-κB activity assay (mean ± SEM, n = 3) in FC1245 cells cultured with CM non-RT CAFs, CM RT-CAFs, and CM RT-CAFs in the presence of 10 μmol/L 1400W. **, P < 0.01, compared with FC1245; &&, P < 0.01 compared with FC1245 treated with CM from non-RT CAFs; $$, P < 0.01, compared with FC1245 treated with CM from RT-CAFs and based on a Student t test. 1400W, N-(3-(aminomethyl)benzyl)acetamidine. F, IFNγ, IL1β, and TNFα 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). Cell supernatants were collected at 24 hours after cell culture and cytokine activity was determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead 32 Plex Panel. Data are presented as mean ± SEM; n = 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, n = 4) of pancreatic orthotopic tumors 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. *, P < 0.05; **, P < 0.01, compared with FC1245. H, Cell growth of FC1245 cultured in the presence or absence of CM of irradiated and nonirradiated CAFs. To deplete iNOS, FC1245 tumor cells were treated with 10 μmol/L 1400W or transfected with iNOS siRNA (iNOS KD). Data are presented as mean ± SEM; n = 3. *, P < 0.05, compared with FC1245 plus CAFs non-RT or FC1245 plus RT-CAFs; &, P < 0.05, compared with FC1245 and based on a Student t test. 1400W, N-(3-(aminomethyl)benzyl)acetamidine. FC1245 migration (I) and invasion (J) after treatment with CM of CAFs using the Boyden and Matrigel invasion chambers, respectively. Data are presented as mean ± SEM, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with FC1245 and based on a Student t test.

Figure 2.

Radiotherapy (RT)-activated CAFs enhance PDAC tumor growth. A, CAFs were isolated from nonradiotherapy-treated FC1245 orthotopic PDACs (non-RT CAFs) or from mice treated with a 12 Gy radiotherapy dose (RT-CAFs). FC1245 were cultured with conditioned medium from CAFs or cocultured with CAFs using a transwell in 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 scanning densitometry with ImageJ software. C, Western blot analysis of iNOS in total lysates of FC1245. FC1245 cells were cocultured with CAFs (RT-CAFs or non-RT CAFs) or treated 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 ImageJ software. 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 compared with FC1245 and based on a Student t test (mean ± SEM, n = 4). E, Luciferase NF-κB activity assay (mean ± SEM, n = 3) in FC1245 cells cultured with CM non-RT CAFs, CM RT-CAFs, and CM RT-CAFs in the presence of 10 μmol/L 1400W. **, P < 0.01, compared with FC1245; &&, P < 0.01 compared with FC1245 treated with CM from non-RT CAFs; $$, P < 0.01, compared with FC1245 treated with CM from RT-CAFs and based on a Student t test. 1400W, N-(3-(aminomethyl)benzyl)acetamidine. F, IFNγ, IL1β, and TNFα 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). Cell supernatants were collected at 24 hours after cell culture and cytokine activity was determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead 32 Plex Panel. Data are presented as mean ± SEM; n = 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, n = 4) of pancreatic orthotopic tumors 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. *, P < 0.05; **, P < 0.01, compared with FC1245. H, Cell growth of FC1245 cultured in the presence or absence of CM of irradiated and nonirradiated CAFs. To deplete iNOS, FC1245 tumor cells were treated with 10 μmol/L 1400W or transfected with iNOS siRNA (iNOS KD). Data are presented as mean ± SEM; n = 3. *, P < 0.05, compared with FC1245 plus CAFs non-RT or FC1245 plus RT-CAFs; &, P < 0.05, compared with FC1245 and based on a Student t test. 1400W, N-(3-(aminomethyl)benzyl)acetamidine. FC1245 migration (I) and invasion (J) after treatment with CM of CAFs using the Boyden and Matrigel invasion chambers, respectively. Data are presented as mean ± SEM, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with FC1245 and based on a Student t test.

Close modal

We next measured nitrite (NO2), a stable byproduct of NO, in the culture medium of CAFs, FC1245 cells, or FC1245 cells cocultured with CAFs (Fig. 2D). Levels of NO2 were higher in CAFs relative to FC1245 cells in both nonirradiated and irradiated experimental conditions (Fig. 2D). These results are consistent with previous studies reporting low amounts of NO in T3M4 and PT45-P1 pancreatic cancer cells (29). Cultures or FC1245 cells with CM of RT-CAFs displayed higher NO2 levels when compared with non-RT CAFs (Fig. 2D). NO2 secretion by FC1245 cells was strongly upregulated by CM of radiotherapy-activated CAFs receiving an extra in vitro radiotherapy dose of 5 Gy (44.7 ± 0.1, mean ± SEM, n = 4; Fig. 2D). Additional studies in SUIT-2, CAPAN-2, MIA PaCa-2, and BxPC3 human pancreatic cancer cell lines cultured with CM of radiotherapy-activated human CAFs demonstrated that an in vitro radiotherapy dose of 5 Gy increases NO2 secretion by cancer cells (Supplementary Fig. S7). These results show that radiotherapy-activated CAFs produce high levels of iNOS and NO secretion, which are further induced by PDAC cells and that radiotherapy enhances iNOS/NO paracrine signaling in PDACs.

Given that inflammatory cytokines such as IFNγ, IL1β, and TNFα induce iNOS expression through NF-κB (22, 29, 30), we performed analyses of NF-κB and 32 cytokines using a MILLIPLEX kit. NF-κB activity in FC1245 cells was monitored using a NF-κB luciferase reporter assay. Culture of FC1245 with CM from RT-CAFs increased NF-κB in the tumor cells (Fig. 2E). Cytokine analyses were performed using the cell supernatant of FC1245, non-RT CAFs, RT-CAFs, and FC1245 cells cultured either with medium from nonradiotherapy fibroblasts or with medium from radiotherapy fibroblasts. IFNγ, IL1β, leukemia-inhibitory factor (LIF), RANTES (regulated on activation normal T-cell expressed and secreted), and IL13 were similar in FC1245 and CAFs (Fig. 2F). Culture of FC1245 PDAC cells in the presence of CM from radiotherapy fibroblasts increased the production of IFNγ, IL1β, and TNFα (Fig. 2F). In addition, when FC1245 cells were cultured with medium from radiotherapy fibroblasts, we observed an increase in the production of IL6, LIF, and RANTES (Supplementary Fig. S8). On the other hand, IL13 decreases when FC1245 cells were cultured with medium from nonradiotherapy fibroblasts (Supplementary Fig. S8). We next used real-time PCR to understand the major genes in radiotherapy-activated CAFs driving these transcriptional differences in FC1245 cancer cells (Supplementary Fig. S9). We observed upregulation in IFNβ, iNOS, and TNFα in RT-CAFs when compared with non-RT CAFs. When compared with RT-CAFs, nonradiotherapy CAFs demonstrated higher mRNA levels for RANTES, C-X-C motif chemokine 10 (CXCL10), IL12, IL1α, IL1β, IL1R1, IL6Rα, Interferon regulatory factor 7 (IRF7), and STAT1 (Supplementary Fig. S9). These results suggest that radiotherapy-activated fibroblasts induce increased secretion of NO and iNOS expression in the tumor cells through NF-κB, which in turn leads to an elevated release of inflammatory cytokines by the tumor cells.

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

Recent studies have demonstrated that PDAC tumor growth in orthotopic murine models is faster in tumors developed by inoculation of tumor cells plus CAFs when compared with tumor cells alone (9). In our study, FC1245 pancreatic orthotopic tumor models were used to determine the role of non-RT CAFs and RT-CAFs in PDAC tumor growth in vivo. FC1245luc+ alone, FC1245luc+ mixed with non-RT CAFs, and FC1245luc+ mixed with RT-CAFs were implanted into the pancreas of C57BL/6 (B6) mice and tumor growth was evaluated using bioluminescence. As shown in Fig. 2G, the tumor growth rate was significantly higher in FC1245 cells mixed with RT-CAFs than that in the model developed by mixing FC1245 cells with non-RT CAFs or FC1245 cells alone (Fig. 2G). At 14 days after cells' implantation in the pancreas, we observed significantly higher αSMA+ in tumors developed by FC1245luc+ mixed with CAFs when compared with FC1245luc+ cells alone (Supplementary Fig. S10). To explain the protumor effect of RT-CAFs, the influence of these cells on the growth of FC1245 cells was investigated in vitro using the Trypan blue assay. Enhanced FC1245 cell growth was observed after cells being cultured with CM of RT-CAFs (Fig. 2H). Culture of FC1245 with CM of nonradiotherapy CAFs did not interfere with PDAC cancer cell growth (Fig. 2H). Next, we used the Boyden chamber (Fig. 2I) and scratch assays (Supplementary Fig. S11) to determine whether RT-CAFs affected the migratory properties of FC1245 cells. Matrigel invasion chamber assays were also used to measure FC1245 invasion properties in the presence of CAFs (Fig. 2J). FC1245 cells in culture with CM of RT-CAFs demonstrated a significant increase in both migration and invasion properties as compared with FC1245 cells alone, which is consistent with previous studies (7). Culture of FC1245 cells with CM of RT-CAFs showed a significantly larger number of migratory and invading cells compared with culture with CM of nonirradiated fibroblasts (Fig. 2I and J; Supplementary Fig. S11). We did not detect metastatic 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 by FC1245luc+ mixed with RT-CAFs (Supplementary Fig. S13).

Premised on our findings (Figs. 1 and 2), we expected that the protumor effects of RT-CAFs are mediated by iNOS/NO signaling. Therefore, we sought to investigate FC1245 cells growth after iNOS amplification or exposure to physiologic levels of the NO donor diethylenetriamine NONOate (DETA-NO). DETA-NO has a half-life of 20 hours and in aqueous solution it spontaneously decomposes to produce a long lasting NO release. Previous studies have reported that DETA-NO increases migration of lung carcinoma cells and proliferation of breast cancer cells (26, 42). Others have reported that low concentrations of DETA-NO (20–2000 nmol/L) induce cancer cell growth, while concentrations higher than 20 μmol/L inhibit cell proliferation (34). In our studies, NO induction in PDAC cells was performed using 1 μmol/L of DETA-NO or with a clustered regularly interspaced short palindromic repeats (CRISPR) activation plasmid (Supplementary Figs. S14 and S15). Treatment of FC1245 pancreatic cancer cells with 1 μmol/L of DETA-NO or with CRISPR activation plasmid increased NO levels and FC1245 cell growth, an effect that is abolished by the addition of the highly selective iNOS inhibitor 1400W (Supplementary Figs. S14 and S15; ref. 43). To support the hypothesis that NO release from CAFs contributes to radiotherapy resistance of pancreatic carcinoma cells, NO induction in PDAC cells in the presence of RT-CAFs was blocked using siRNA-mediated knockdown of iNOS or pharmacologic treatments with 1400W (Fig. 2H; Supplementary Figs. S14–S16). In addition, the iNOS inhibitor 1400W decreased NF-κB induction in tumor cells cultured with CM RT-CAFs (Fig. 2E). These results indicate that CAFs can promote the growth of PDAC cells, an effect that increases when cells are cultured with CM of RT-CAFs, which can be abolished in vitro by iNOS depletion using genetic and pharmacologic approaches.

Pharmacologic blockade of iNOS improves therapeutic response of PDACs to radiotherapy

Previous studies demonstrated a role for iNOS in PDAC chemoresistance (20). Our findings suggest iNOS/NO signaling as a cause of PDAC resistance to radiotherapy, with NO secretion from RT-CAFs preventing radiotherapy therapeutic efficacy. To translate our findings into a clinically relevant approach, we performed therapeutic studies combining iNOS pharmacologic inhibition with radiotherapy in orthotopic PDAC models. Luciferase-expressing FC1245 pancreatic cancer cells were implanted into the pancreas of C57BL/6 (B6) mice. To determine the impact of radiotherapy dose on PDAC therapy, a pilot therapeutic study was performed using 12 Gy in a single treatment fraction or 18 Gy administered in three fractions of 6 Gy at 48-hour intervals. These dose schedules were selected because, as per the linear quadratic model of cell kill, they have similar biologically effective dose (BED at an alpha/beta = 10, BED10). The three-fraction course of radiotherapy (BED10 = 28.8 Gy) improved survival when compared with a single fraction (BED10 = 26.4 Gy; Supplementary Fig. S17), supporting previous reports that are suggestive of the clinical benefit of radiotherapy administration in fractions (11). In further therapeutic experiments, radiotherapy was administered in 3 doses of 6 Gy at 48-hour intervals. After an engraftment period, tumor-bearing mice as identified via bioluminescence were randomly assigned to treatment groups (nonradiotherapy, radiotherapy, 1400W, and 1400W/radiotherapy). At 14 days after the first radiotherapy dose, mice treated with 1400W or 1400W/radiotherapy demonstrated significantly decreased tumor growth and tumors of small size as measured by luminescence (Fig. 3AC). The weight of tumors at 14 days after the first radiotherapy dose was 1.65 ± 0.60, 0.92 ± 0.04, and 0.60 ± 0.09 g in mice treated with radiotherapy, 1400W, and 1400W/radiotherapy (Supplementary Fig. S18).

Figure 3.

Pharmacologic blockade of iNOS improves therapeutic response of PDACs to radiotherapy. A-C, FC1245 luciferase–expressing PDAC orthotopic tumors treated with radiotherapy, 1400W, or 1400W/radiotherapy (n = 10 mice per group) and tracked by bioluminescence. Mice were treated with 3 doses of 6 Gy at 48-hour intervals. Intraperitoneal administration of 1400W (1 μg/g) was started at day 0. 1400W was administered every day for 7 days during the radiotherapy 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 (n = 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 = 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 with radiotherapy; $, P < 0.05; $$, P < 0.01, compared with 1400W and based on a Student t test.

Figure 3.

Pharmacologic blockade of iNOS improves therapeutic response of PDACs to radiotherapy. A-C, FC1245 luciferase–expressing PDAC orthotopic tumors treated with radiotherapy, 1400W, or 1400W/radiotherapy (n = 10 mice per group) and tracked by bioluminescence. Mice were treated with 3 doses of 6 Gy at 48-hour intervals. Intraperitoneal administration of 1400W (1 μg/g) was started at day 0. 1400W was administered every day for 7 days during the radiotherapy 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 (n = 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 = 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 with radiotherapy; $, P < 0.05; $$, P < 0.01, compared with 1400W and based on a Student t test.

Close modal

One of the downstream effects of NO in cancer includes evasion to apoptosis (24, 44) and therefore, tumor sections of the different therapeutic groups were immunostained to assess proliferation and apoptosis. The 1400W/radiotherapy-treated mice displayed diminished proliferation of tumor cells based on the reduced number of Ki67+ tumor cells compared with control, 1400W, and radiotherapy groups (Fig. 3D). IHC staining of cleaved caspase-3 indicated significantly increased apoptosis in pancreatic tumors treated with radiotherapy and 1400W/radiotherapy (Fig. 3D). Collectively, these results demonstrate that radiotherapy/iNOS inhibition slows pancreatic tumor growth.

NO secretion from RT-CAFs changes PDAC TME extracellular pH

One of the mechanisms by which NO modulates tumor cell proliferation is through upregulation of glycolysis (34). NO release into tumor cells reduces mitochondrial oxidative phosphorylation at complexes III and IV of the electron transport chain and increases activity of the 6-phosphofructokinase 1 (PFK-1), which results in the activation of glycolysis promoting a metabolic switch from an aerobic to a glycolytic phenotype (Warburg effect; refs. 34, 45–50). We attempted to study changes in pancreatic cancer cells glycolysis after FC1245 cells incubation with the NO-donor DETA-NO or culture with CM of RT-CAFs. Our results show that NO production in FC1245 cells is increased through exposure to DETA-NO (Supplementary Fig. S14). Glucose consumption and lactate production (Supplementary Fig. S19) are increased compared with without DETA-NO addition. This effect was further inhibited by the addition of the iNOS inhibitor 1400W (Supplementary Fig. S19).

Because our results showed that DETA-NO increased the glycolytic rate when compared with untreated FC1245 cells, we hypothesized that NO release from CAFs could increase glucose consumption and lactate secretion in cancer cells. Cultures of FC1245 with CM of CAFs demonstrated that RT-CAFs led to a significant increase in glucose uptake (1.3 ± 0.1, mean ± SEM, n = 4; Fig. 4A) and lactate production (2.1 ± 0.6, mean ± SEM, n = 4; Fig. 4A). At 14 days postradiotherapy, we observed an increase in GLUT1 expression of PDAC tumors (1.7 ± 0.2, mean ± SEM, n = 3; Fig. 4B). Additional immunofluorescence staining of αSMA, cytokeratin, and GLUT1 in radiotherapy-treated PDAC samples demonstrated GLUT1+/αSMA+ in 8.4% of total αSMA+ cells and GLUT1+/cytokeratin+ in 34.3% of total cytokeratin+ cells (Fig. 4C and D; Supplementary Tables S8–S11). The percentage of GLUT1+/cytokeratin+ cells was 7.6-fold higher in radiotherapy-treated samples when compared with control (Fig. 4C and D; Supplementary Tables S8–S11). We next used fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) to validate in vivo changes in GLUT1 and glucose consumption observed in Fig. 4AD. Our biodistribution and PET imaging results at 14 days post first radiotherapy dose in mice bearing FC1245 orthotopic PDAC tumors demonstrated a significant increase in 18F-FDG PDAC uptake, an effect that decreases in mice treated with radiotherapy/1400W (Fig. 4E and F; Supplementary Fig. S20). As glycolysis promotes intracellular H+ generation contributing to the membrane dynamics of Na+/H+ flux, as mediated by the sodium/hydrogen exchanger isoform 1 (NHE1), an increase in glycolysis as mediated by NO release from RT-CAFs could activate NHE1. Our results revealed that radiotherapy increases NHE1 expression in FC1245 PDACs, an effect that was inhibited with the iNOS inhibitor 1400W (Fig. 4G). In sum, our findings indicate that an increase in iNOS/NO signaling in RT-fibroblasts upregulates PDAC glycolysis.

Figure 4.

Radiotherapy-activated CAFs change PDAC-TME extracellular pH, allowing detection with pH-targeted pHLIP. A, Glucose uptake and lactate production of FC1245 PDAC cells treated with CM from CAFs. Data are presented as mean ± SEM, n = 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/αSMA (C) and GLUT1/cytokeratin (D) in total lysates of FC1245 orthotopic PDAC tumors obtained at 14 days postradiotherapy. Density of Western blot bands was quantified by scanning densitometry 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 mice bearing FC1245 orthotopic tumors. At 14 days posttherapy, mice were administered 200 μCi intravenous injection of 18F-FDG. Bars, n = 4 mice per group; mean ± SEM. %ID, percentage of injected dose. G, Confocal images and quantification (mean ± SEM, n = 3) of immunofluorescence staining of NHE1 (green) in FC1245 orthotopic PDAC tumors obtained at 14 days postradiotherapy. DAPI (blue) was used to stain cell nuclei. Scale bars, 50 μm. H and I, PDAC tumor uptake as determined by acute biodistribution studies at 24 hours after injection of 67Ga-labeled pHLIP in mice bearing FC1245 orthotopic tumors. At 14 days postradiotherapy, mice were intravenously given 20 μCi of 67Ga-labeled pHLIP. Bars, n = 4 mice per group; mean ± SEM. %ID/g, percentage of injected dose per gram. J, An increase in glycolysis 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 more alkaline 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.

Figure 4.

Radiotherapy-activated CAFs change PDAC-TME extracellular pH, allowing detection with pH-targeted pHLIP. A, Glucose uptake and lactate production of FC1245 PDAC cells treated with CM from CAFs. Data are presented as mean ± SEM, n = 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/αSMA (C) and GLUT1/cytokeratin (D) in total lysates of FC1245 orthotopic PDAC tumors obtained at 14 days postradiotherapy. Density of Western blot bands was quantified by scanning densitometry 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 mice bearing FC1245 orthotopic tumors. At 14 days posttherapy, mice were administered 200 μCi intravenous injection of 18F-FDG. Bars, n = 4 mice per group; mean ± SEM. %ID, percentage of injected dose. G, Confocal images and quantification (mean ± SEM, n = 3) of immunofluorescence staining of NHE1 (green) in FC1245 orthotopic PDAC tumors obtained at 14 days postradiotherapy. DAPI (blue) was used to stain cell nuclei. Scale bars, 50 μm. H and I, PDAC tumor uptake as determined by acute biodistribution studies at 24 hours after injection of 67Ga-labeled pHLIP in mice bearing FC1245 orthotopic tumors. At 14 days postradiotherapy, mice were intravenously given 20 μCi of 67Ga-labeled pHLIP. Bars, n = 4 mice per group; mean ± SEM. %ID/g, percentage of injected dose per gram. J, An increase in glycolysis 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 more alkaline 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.

Close modal

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

Activation of NHE1 causes a reversal of the plasma membrane pH gradient (51), resulting in a more alkaline intracellular pH and a more acidic extracellular pH (Fig. 4HJ). As we observed an increase in GLUT1 and NHE1 in radiotherapy-treated tumors (Fig. 4AG), we attempted to use a pH-targeted imaging probe to detect changes in the extracellular microenvironment pH, as mediated 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 the radiometallated pHLIP variant [67Ga]Ga-NO2A-cysVar3 in mice bearing FC1245 orthotopic PDAC tumors. On the basis of our studies demonstrating that at 14 days posttherapy, the tumor growth was slowed in mice treated with 1400W/radiotherapy when compared with radiotherapy alone (Fig. 3AC), mice were administered with [67Ga]Ga-NO2A-cysVar3 at 14 days post first radiotherapy dose. At 24 hours postinjection of [67Ga]Ga-NO2A-cysVar3, PDAC radiotherapy-treated tumors had an uptake of 8.6 ± 0.7 percentage injected dose per gram tissue (%ID/g, mean ± SEM, n = 5), while control tumors had an uptake of 3.6% ± 1.9% ID/g, mean ± SEM, n = 5 (Fig. 4I; Supplementary Fig. S21). The increase in [67Ga]Ga-NO2A-cysVar3 PDAC tumor uptake, due to an increase in extracellular acidity, was further decreased in mice treated with radiotherapy plus 1400W (3.1 ID/g ± 1.0, mean ± SEM, n = 5, Fig. 4I). Taken together, these results suggest that NO signaling activation in radiotherapy-treated tumors will result in changes of the PDAC extracellular pH (Fig. 4J) that can be detected using radiolabeled pHLIP, suggesting a clinical potential of pHLIP to assess radiotherapy response within pancreatic cancer, and suggesting ultimately that the benefit of radiotherapy intervention in PDAC can be improved by blocking NO signaling.

The mechanisms 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 and subsequent NO secretion in PDAC. Mechanistic studies demonstrated that the CM of irradiated CAFs drives the production of iNOS in PDACs. Cultures of PDAC cells with CM of RT-CAFs demonstrate that NO secretion and iNOS expression by RT-CAFs are further induced by pancreatic tumor cells through NF-κB, which in turn leads to the release of inflammatory cytokines by the tumor cells. In addition, we found that NO increases after radiotherapy in PDAC, results in acidification of the microenvironment, which can be detected by molecular imaging using pHLIP. These data support that NO has a protumor function in irradiated PDACs by promoting a glycolytic phenotype as detected in vivo by 18F-FDG PET imaging, which leads to extracellular acidosis. iNOS pharmacologic inhibition or knockdown improved radiotherapy efficacy and decreased PDAC tumor growth, suggesting an integral role for NO and radiotherapy-mediated iNOS activity 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 or NOS3). 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 transcriptionally regulated and induced by inflammatory cytokines (21, 22, 29). The expression of iNOS and production of NO have been detected in several tumor types and are comprehensively reviewed elsewhere (22). Our findings show that, in contrast to PDAC samples, nontumor tissues exhibit lower iNOS expression levels (Figs. 1 and 2; refs. 20, 28). Previous studies have demonstrated that iNOS expression is found not only in the tumor, but also in CAFs, inflammatory, and endothelial cells (29). Further studies are necessary to evaluate the consequences of radiotherapy on NOS expression in other noncancer cells that contribute to the stromal compartment of the PDAC TME.

While some studies have demonstrated that tumor cell–derived NO inhibits tumor progression (22, 23, 26, 42, 53, 54), others have demonstrated its role in the development of tumor growth (22, 55, 56). Similar to what has been observed for other tumor types, the role NO plays in PDAC is also not clearly defined. PDAC tumor cells containing low levels of NO exhibit an aggressive metastatic phenotype when compared with tumor cells containing high NO levels (21) and treatment with a NO-donor inhibited proliferation and invasion of PDAC (54). Other studies have reported an association between high iNOS expression and poorer survival in patients with PDAC (20) and a role for iNOS/NO signaling in PDAC chemoresistance (29). Conversely, low iNOS levels are associated with enhanced survival in KPC mice containing orthotopic PDAC (20). Our studies clearly establish that radiotherapy increases iNOS expression and NO signaling in PDAC contributing to tumor resistance to therapy.

The contribution of NO 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 through alterations in inflammatory secretome (6). To determine the cellular sources responsible for iNOS/NO increase in PDAC after radiotherapy, we performed in vitro studies using pancreatic tumor cells in culture with CAFs in a transwell coculture system or with CM of these fibroblasts. Our data show that, although undetectable in radiotherapy-activated tumor cells, RT-CAFs showed significant iNOS expression and NO secretion (Fig. 2BD). In our experiments, an extra in vitro radiotherapy dose was necessary to study the mechanisms by which RT-CAFs induce iNOS/NO secretion in the tumors cells (Fig. 2). When compared with non-RT-CAFs, RT-CAFs demonstrated upregulation in IFNβ, iNOS, and TNFα (Supplementary Fig. S9). Coculture of PDAC cells with RT-CAFs and culture with CM of CAFs increased iNOS expression/NO secretion in the tumors cells (Fig. 2C and D). The increase in iNOS expression and NO secretion by the tumor cells was higher when cells were cultured with CAF-CM when compared with cells cocultured with CAFs (Fig. 2C and D). These results suggest that the secretome of RT-CAFs is the major driving force in the production of iNOS in PDACs. The differences in iNOS/NO between FC1245 cocultures and cultures with CAF-CM can be explained by changes in PDAC cells viability (Supplementary Fig. S6) and future studies are necessary to determine the temporal dynamics of iNOS/NO signaling in these in vitro culture systems.

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

Because NO regulates several biochemical pathways, the molecular mechanisms by which NO increases in CAFs after radiotherapy, and how iNOS/NO signaling can be further induced by the tumor cells facilitating tumor growth (Fig. 2E) are likely broad. At the transcriptional level, TNFα, IFNγ, IL6, and IL1β are all known to activate NF-κB, which induces iNOS expression (22, 29, 30). Here, we show that the protumor inducing effect of radiotherapy-activated fibroblasts occurs through NF-κB and involves increased secretion of IFNγ, IL1β, TNFα, IL6, and LIF (an IL6 class cytokine) by pancreatic tumor cells. In addition, our studies demonstrate that the iNOS inhibitor 1400W decreases NF-κB induction in PDACs cultured with secretome from RT-CAFs.

Preclinical and clinical studies have shown low toxicity of iNOS inhibitors (33, 57–59), suggesting their potential as therapeutic agents. For instance, iNOS inhibitors have been show to block colon adenocarcinoma (60) and glioma tumor stem cell tumor growth (33). Our findings that radiotherapy increases iNOS expression, suggests iNOS inhibition as a promising strategy that could be applied in the treatment of PDAC. In our studies, the iNOS inhibitor 1400W delayed tumor growth, an effect that was enhanced with the 1400W/radiotherapy combination therapy (Fig. 3AC). Importantly, the 1400W/radiotherapy therapeutic approach shows potential to increase preservation of healthy tissues during surgical PDAC interventions due to the observation of increasingly localized tumors with less diffuse margins (Fig. 3B). The 1400W/radiotherapy combination therapy was unable to completely eliminate tumor growth and further studies are necessary to determine whether other iNOS inhibitors or combination of iNOS inhibition with other treatment regimens may further augment efficacy when compared with 1400W/radiotherapy. Previous reports in orthotopic intracranial tumors demonstrated higher therapeutic efficacy for the iNOS inhibitor BYK191023 when compared with 1400W (33) plausibly due to its improved pharmacokinetic and bioavailability properties.

In ovarian cancer cells, NO decreases mitochondrial respiration, which results in the activation of glycolysis promoting a metabolic switch from an aerobic to a glycolytic phenotype (34). Glycolysis promotes H+ generation, which is removed from the intracellular to the extracellular compartments in exchange with Na+, by NHE1 (51). In our studies, we observed that NO increases glycolytic rate and NHE1 expression in PDAC (Fig. 4AG). These membrane transport mechanisms 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 extracellular pH arising from radiotherapy-mediated NO secretion (Fig. 4H and I). The var3 pHLIP was used in our studies due to its remarkable ability to target acidity in vivo (52, 61–63) and a radiolabeled version of var3 pHLIP will be entering in the clinic in the current year of 2020. The radiolabeled var3 pHLIP demonstrated higher targeting accumulation in radiotherapy PDAC tumors when compared with unirradiated PDAC tumors (Fig. 4I). This increase in acidity, due to NO secretion from radiotherapy-activated PDACs, was further decreased when radiotherapy is combined with the iNOS inhibitor 1400W.

In conclusion, our data support the notion that the secretome of RT-CAFs initiates a paracrine activation loop increasing iNOS/NO signaling in PDAC and additional in vitro radiation is necessary for robust iNOS expression by the tumor cells. Our preclinical results demonstrate the potential of iNOS inhibition with fractionated radiotherapy regimen for improved response, and additionally that that response can be assessed via radiolabeled pHLIP, highlighting the mechanisms by which NO in the secretome of RT-CAFs contributes to PDAC resistance. Our study supports previous reports demonstrating that the secretome of therapy-activated CAFs contribute to PDAC pathology and that targeting iNOS/NO signaling may be a valid therapeutic approach for improving radiotherapy outcomes in PDAC.

T. 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, Leap Therapeutics, and Aprea and has ownership interest (including patents) in oncolytic viral 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.

Conception and design: P.M.R. Pereira, T. Merghoub, J.S. Lewis

Development of methodology: P.M.R. Pereira, K.J. Edwards, F.E. Escorcia

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.M.R. Pereira, K. Mandleywala, L.M. Carter, L.F. Campesato, M. Cornejo, L. Abma, A.-A. Mohsen, J.S. Lewis

Analysis 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. Lewis

Writing, 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. Lewis

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P.M.R. Pereira, J.S. Lewis

Study supervision: J.S. Lewis

The authors acknowledge the Radiochemistry and Molecular Imaging Probe Core, the Antitumor Assessment Core, and Molecular Cytology Core Facility, which were supported by NIH grant P30 CA08748. This study was supported in part by the Geoffrey Beene Cancer Research Center of MSKCC (to J.S. Lewis) and NIH NCI R35 CA232130 (to J.S. Lewis). We gratefully acknowledge Mr. William H. and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research and The Center for Experimental Therapeutics of MSKCC. P.M.R. Pereira acknowledges the Tow Foundation Postdoctoral Fellowship from the MSKCC Center for Molecular Imaging and Nanotechnology and the Alan and Sandra Gerry Metastasis and Tumor Ecosystems 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 Across America, Ludwig Institute for Cancer Research, Ludwig Center for Cancer Immunotherapy at Memorial Sloan Kettering, Cancer Research Institute, Parker Institute for Cancer Immunotherapy and Breast Cancer Research Foundation. Figures 2A, 4H, and 4J were created with Biorender. We thank Dr. Russel and Dr. Monette for their help with the Arc Therapy and image analysis, respectively. We thank Egger for cutting the human samples used in this work. We thank Carl DeSelm in the Michel Sadelain lab for generating the FC1245luc+ PDAC cells.

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

1.
Siegel
RL
,
Miller
KD
,
Jemal
A
. 
Cancer statistics
, 
2019
.
CA Cancer J Clin
2019
;
69
:
7
34
.
2.
Kleeff
J
,
Korc
M
,
Apte
M
,
La Vecchia
C
,
Johnson
CD
,
Biankin
AV
, et al
Pancreatic cancer
.
Nat Rev Dis Primers
2016
;
2
:
16022
.
3.
Conlon
KC
,
Klimstra
DS
,
Brennan
MF
. 
Long-term survival after curative resection for pancreatic ductal adenocarcinoma. Clinicopathologic analysis of 5-year survivors
.
Ann Surg
1996
;
223
:
273
9
.
4.
Neoptolemos
JP
,
Stocken
DD
,
Friess
H
,
Bassi
C
,
Dunn
JA
,
Hickey
H
, et al
A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer
.
N Engl J Med
2004
;
350
:
1200
10
.
5.
Bhide
SA
,
Nutting
CM
. 
Recent advances in radiotherapy
.
Bmc Med
2010
;
8
:
25
.
6.
Tommelein
J
,
De Vlieghere
E
,
Verset
L
,
Melsens
E
,
Leenders
J
,
Descamps
B
, et al
Radiotherapy-activated cancer-associated fibroblasts promote tumor progression through paracrine IGF1R activation
.
Cancer Res
2018
;
78
:
659
70
.
7.
Ohuchida
K
,
Mizumoto
K
,
Murakami
M
,
Qian
LW
,
Sato
N
,
Nagai
E
, et al
Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interactions
.
Cancer Res
2004
;
64
:
3215
22
.
8.
Hellevik
T
,
Pettersen
I
,
Berg
V
,
Bruun
J
,
Bartnes
K
,
Busund
LT
, et al
Changes in the secretory profile of NSCLC-associated fibroblasts after ablative radiotherapy: potential impact on angiogenesis and tumor growth
.
Transl Oncol
2013
;
6
:
66
74
.
9.
Ligorio
M
,
Sil
S
,
Malagon-Lopez
J
,
Nieman
LT
,
Misale
S
,
Di Pilato
M
, et al
Stromal microenvironment shapes the intratumoral architecture of pancreatic cancer
.
Cell
2019
;
178
:
160
75
.
10.
Gukovsky
I
,
Li
N
,
Todoric
J
,
Gukovskaya
A
,
Karin
M
. 
Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer
.
Gastroenterology
2013
;
144
:
1199
209
.
11.
Seifert
L
,
Werba
G
,
Tiwari
S
,
Giao Ly
NN
,
Nguy
S
,
Alothman
S
, et al
Radiation therapy induces macrophages to suppress T-cell responses against pancreatic tumors in mice
.
Gastroenterology
2016
;
150
:
1659
72
.
12.
Hallahan
DE
,
Spriggs
DR
,
Beckett
MA
,
Kufe
DW
,
Weichselbaum
RR
. 
Increased tumor necrosis factor-alpha mRNA after cellular exposure to ionizing radiation
.
Proc Natl Acad Sci U S A
1989
;
86
:
10104
7
.
13.
Burnette
BC
,
Liang
H
,
Lee
Y
,
Chlewicki
L
,
Khodarev
NN
,
Weichselbaum
RR
, et al
The efficacy of radiotherapy relies upon induction of type I interferon-dependent innate and adaptive immunity
.
Cancer Res
2011
;
71
:
2488
96
.
14.
Lugade
AA
,
Sorensen
EW
,
Gerber
SA
,
Moran
JP
,
Frelinger
JG
,
Lord
EM
. 
Radiation-induced IFN-gamma production within the tumor microenvironment influences antitumor immunity
.
J Immunol
2008
;
180
:
3132
9
.
15.
Mantoni
TS
,
Lunardi
S
,
Al-Assar
O
,
Masamune
A
,
Brunner
TB
. 
Pancreatic stellate cells radioprotect pancreatic cancer cells through β1-integrin signaling
.
Cancer Res
2011
;
71
:
3453
8
.
16.
Hellevik
T
,
Martinez-Zubiaurre
I
. 
Radiotherapy and the tumor stroma: the importance of dose and fractionation
.
Front Oncol
2014
;
4
:
1
.
17.
Lagares-Garcia
JA
,
Moore
RA
,
Collier
B
,
Heggere
M
,
Diaz
F
,
Qian
F
. 
Nitric oxide synthase as a marker in colorectal carcinoma
.
Am Surg
2001
;
67
:
709
13
.
18.
Ekmekcioglu
S
,
Ellerhorst
JA
,
Prieto
VG
,
Johnson
MM
,
Broemeling
LD
,
Grimm
EA
. 
Tumor iNOS predicts poor survival for stage III melanoma patients
.
Int J Cancer
2006
;
119
:
861
6
.
19.
Glynn
SA
,
Boersma
BJ
,
Dorsey
TH
,
Yi
M
,
Yfantis
HG
,
Ridnour
LA
, et al
Increased NOS2 predicts poor survival in estrogen receptor-negative breast cancer patients
.
J Clin Invest
2010
;
120
:
3843
54
.
20.
Wang
J
,
He
PJ
,
Gaida
M
,
Yang
S
,
Schetter
AJ
,
Gaedcke
J
, et al
Inducible nitric oxide synthase enhances disease aggressiveness in pancreatic cancer
.
Oncotarget
2016
;
7
:
52993
3004
.
21.
Wang
B
,
Wei
DY
,
Crum
VE
,
Richardson
EL
,
Xiong
HH
,
Luo
Y
, et al
A novel model system for studying the double-edged roles of nitric oxide production in pancreatic cancer growth and metastasis
.
Oncogene
2003
;
22
:
1771
82
.
22.
Fukumura
D
,
Kashiwagi
S
,
Jain
RK
. 
The role of nitric oxide in tumour progression
.
Nat Rev Cancer
2006
;
6
:
521
34
.
23.
Bailey
P
,
Chang
DK
,
Forget
MA
,
Lucas
FA
,
Alvarez
HA
,
Haymaker
C
, et al
Exploiting the neoantigen landscape for immunotherapy of pancreatic ductal adenocarcinoma
.
Sci Rep
2016
;
6
:
35848
.
24.
Engels
K
,
Knauer
SK
,
Loibl
S
,
Fetz
V
,
Harter
P
,
Schweitzer
A
, et al
NO signaling confers cytoprotectivity through the survivin network in ovarian carcinomas
.
Cancer Res
2008
;
68
:
5159
66
.
25.
Rieder
J
,
Jahnke
R
,
Schloesser
M
,
Seibel
M
,
Czechowski
M
,
Marth
C
, et al
Nitric oxide-dependent apoptosis in ovarian carcinoma cell lines
.
Gynecol Oncol
2001
;
82
:
172
6
.
26.
Pervin
S
,
Singh
R
,
Hernandez
E
,
Wu
GY
,
Chaudhuri
G
. 
Nitric oxide in physiologic concentrations targets the translational machinery to increase the proliferation of human breast cancer cells: involvement of mammalian target of Rapamycin/eIF4E pathway
.
Cancer Res
2007
;
67
:
289
99
.
27.
Kisley
LR
,
Barrett
BS
,
Bauer
AK
,
Dwyer-Nield
LD
,
Barthel
B
,
Meyer
AM
, et al
Genetic ablation of inducible nitric oxide synthase decreases mouse lung tumorigenesis
.
Cancer Res
2002
;
62
:
6850
6
.
28.
Vickers
SM
,
MacMillan-Crow
LA
,
Green
M
,
Ellis
C
,
Thompson
JA
. 
Association of increased immunostaining for inducible nitric oxide synthase and nitrotyrosine with fibroblast growth factor transformation in pancreatic cancer
.
Arch Surg
1999
;
134
:
245
51
.
29.
Müerköster
S
,
Wegehenkel
K
,
Arlt
A
,
Witt
M
,
Sipos
B
,
Kruse
ML
, et al
Tumor stroma interactions induce chemoresistance in pancreatic ductal carcinoma cells involving increased secretion and paracrine effects of nitric oxide and interleukin-1β
.
Cancer Res
2004
;
64
:
1331
7
.
30.
Arlt
A
,
Vorndamm
J
,
Muerkoster
S
,
Yu
H
,
Schmidt
WE
,
Fölsch
UR
, et al
Autocrine production of interleukin-1-β confers constitutive NFκB activity and chemoresistance in pancreatic carcinoma cell lines
.
Cancer Res
2002
;
62
:
910
6
.
31.
Khalili
JS
,
Liu
SJ
,
Rodriguez-Cruz
TG
,
Whittington
M
,
Wardell
S
,
Liu
C
, et al
Oncogenic BRAF(V600E) promotes stromal cell-mediated immunosuppression via induction of interleukin-1 in melanoma
.
Clin Cancer Res
2012
;
18
:
5329
40
.
32.
Du
Q
,
Zhang
X
,
Liu
Q
,
Zhang
X
,
Bartels
CE
,
Geller
DA
. 
Nitric oxide production upregulates Wnt/β-catenin signaling by inhibiting Dickkopf-1
.
Cancer Res
2013
;
73
:
6526
37
.
33.
Eyler
CE
,
Wu
QL
,
Yan
K
,
MacSwords
JM
,
Chandler-Militello
D
,
Misuraca
KL
, et al
Glioma stem cell proliferation and tumor growth are promoted by nitric oxide synthase-2
.
Cell
2011
;
146
:
53
66
.
34.
Caneba
CA
,
Yang
L
,
Baddour
J
,
Curtis
R
,
Win
J
,
Hartig
S
, et al
Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells
.
Cell Death Dis
2014
;
5
:
e1302
.
35.
DeSelm
C
,
Palomba
ML
,
Yahalom
J
,
Hamieh
M
,
Eyquem
J
,
Rajasekhar
VK
, et al
Low-dose radiation conditioning enables CAR T cells to mitigate antigen escape
.
Mol Ther
2018
;
26
:
2542
52
.
36.
Escorcia
FE
,
Houghton
JL
,
Abdel-Atti
D
,
Pereira
PR
,
Cho
A
,
Gutsche
NT
, et al
ImmunoPET predicts response to Met-targeted radioligand therapy in models of pancreatic cancer resistant to Met kinase inhibitors
.
Theranostics
2019
;
10
:
151
65
.
37.
Thorek
D. LJ
,
Kramer
RM
,
Chen
Q
,
Jeong
J
,
Lupu
ME
,
Lee
AM
, et al
Reverse-contrast imaging and targeted radiation therapy of advanced pancreatic cancer models
.
Int J Radiat Oncol Biol Phys
2015
;
93
:
444
53
.
38.
Hruban
RH
,
Adsay
NV
,
Albores-Saavedra
J
,
Anver
MR
,
Biankin
AV
,
Boivin
GP
, et al
Pathology of genetically engineered mouse models of pancreatic exocrine cancer: Consensus report and recommendations
.
Cancer Res
2006
;
66
:
95
106
.
39.
Hingorani
SR
,
Wang
L
,
Multani
AS
,
Combs
C
,
Deramaudt
TB
,
Hruban
RH
, et al
Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice
.
Cancer Cell
2005
;
7
:
469
83
.
40.
Erkan
M
,
Michalski
CW
,
Rieder
S
,
Reiser-Erkan
C
,
Abiatari
I
,
Kolb
A
, et al
The activated stroma index is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma
.
Clin Gastroenterol Hepatol
2008
;
6
:
1155
61
.
41.
Erkan
M
,
Hausmann
S
,
Michalski
CW
,
Fingerle
AA
,
Dobritz
M
,
Kleeff
J
, et al
The role of stroma in pancreatic cancer: diagnostic and therapeutic implications
.
Nat Rev Gastroenterol Hepatol
2012
;
9
:
454
67
.
42.
Sanuphan
A
,
Chunhacha
P
,
Pongrakhananon
V
,
Chanvorachote
P
. 
Long-term nitric oxide exposure enhances lung cancer cell migration
.
Biomed Res Int
2013
.
DOI: 10.1155/2013/186972
.
43.
Garvey
EP
,
Oplinger
JA
,
Furfine
ES
,
Kiff
RJ
,
Laszlo
F
,
Whittle
BJ
, et al
1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo
.
J Biol Chem
1997
;
272
:
4959
63
.
44.
Levesque
MC
,
Misukonis
MA
,
O'Loughlin
CW
,
Chen
Y
,
Beasley
BE
,
Wilson
DL
, et al
IL-4 and interferon gamma regulate expression of inducible nitric oxide synthase in chronic lymphocytic leukemia cells
.
Leukemia
2003
;
17
:
442
50
.
45.
Sanhueza
C
,
Araos
J
,
Naranjo
L
,
Sáez
T
,
Silva
L
,
Salsoso
R
, et al
NHE1 promote cell proliferation in ovarian cancer: a role of hypoxia-inducible factors
.
Int J Gynecol Cancer
2015
;
25
:
55
6
.
46.
Sanhueza
C
,
Araos
J
,
Naranjo
L
,
Villalobos
R
,
Westermeier
F
,
Salomon
C
, et al
Modulation of intracellular pH in human ovarian cancer
.
Curr Mol Med
2016
;
16
:
23
32
.
47.
Spugnini
EP
,
Sonveaux
P
,
Stock
C
,
Perez-Sayans
M
,
De Milito
A
,
Avnet
S
, et al
Proton channels and exchangers in cancer
.
Bba-Biomembranes
2015
;
1848
:
2715
26
.
48.
Granados-Principal
S
,
Liu
Y
,
Guevara
ML
,
Blanco
E
,
Choi
DS
,
Qian
W
, et al
Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer
.
Breast Cancer Res
2015
;
17
:
25
.
49.
Harhaji
L
,
Popadic
D
,
Miljkovic
D
,
Cvetkovic
I
,
Isakovic
A
,
Trajkovic
V
. 
Acidosis affects tumor cell survival through modulation of nitric oxide release
.
Free Radic Biol Med
2006
;
40
:
226
235
.
50.
Riemann
A
,
Ihling
A
,
Thomas
J
,
Schneider
B
,
Thews
O
,
Gekle
M
. 
Acidic environment activates inflammatory programs in fibroblasts via a cAMP-MAPK pathway
.
Bba-Mol Cell Res
2015
;
1853
:
299
307
.
51.
Orlowski
J
,
Grinstein
S
. 
Diversity of the mammalian sodium/proton exchanger SLC9 gene family
.
Pflügers Arch Eur J Physiol
2004
;
447
:
549
65
.
52.
Demoin
DW
,
Wyatt
LC
,
Edwards
KJ
,
Abdel-Atti
D
,
Sarparanta
M
,
Pourat
J
, et al
PET Imaging of extracellular pH in tumors with 64Cu- and 18F-labeled pHLIP peptides: a structure-activity optimization study
.
Bioconjug Chem
2016
;
27
:
2014
23
.
53.
Barreiro Arcos
ML
,
Gorelik
G
,
Klecha
A
,
Goren
N
,
Cerquetti
C
,
Cremaschi
GA
. 
Inducible nitric oxide synthase-mediated proliferation of a T lymphoma cell line
.
Nitric Oxide
2003
;
8
:
111
8
.
54.
Sugita
H
,
Kaneki
M
,
Furuhashi
S
,
Hirota
M
,
Takamori
H
,
Baba
H
. 
Nitric oxide inhibits the proliferation and invasion of pancreatic cancer cells through degradation of insulin receptor substrate-1 protein
.
Mol Cancer Res
2010
;
8
:
1152
63
.
55.
Le
XD
,
Wei
DY
,
Huang
SY
,
Lancaster
JR
,
Xie
KP
. 
Nitric oxide synthase II suppresses the growth and metastasis of human cancer regardless of its up-regulation of protumor factors
.
Proc Natl Acad Sci U S A
2005
;
102
:
8758
63
.
56.
Nunokawa
Y
,
Tanaka
S.
Interferon-γ inhibits proliferation of rat vascular smooth-muscle cells by nitric-oxide generation
.
Biochem Bioph Res Commun
1992
;
188
:
409
15
.
57.
Brindicci
C
,
Ito
K
,
Torre
O
,
Barnes
PJ
,
Kharitonov
SA
. 
Effects of aminoguanidine, an inhibitor of inducible nitric oxide synthase, on nitric oxide production and its metabolites in healthy control subjects, healthy smokers, and COPD patients
.
Chest
2009
;
135
:
353
67
.
58.
Dover
AR
,
Chia
S
,
Ferguson
JW
,
Megson
IL
,
Fox
KA
,
Newby
DE
. 
Inducible nitric oxide synthase activity does not contribute to the maintenance of peripheral vascular tone in patients with heart failure
.
Clin Sci
2006
;
111
:
275
80
.
59.
Singh
D
,
Richards
D
,
Knowles
RG
,
Schwartz
S
,
Woodcock
A
,
Langley
S
, et al
Selective inducible nitric oxide synthase inhibition has no effect on allergen challenge in asthma
.
Am J Resp Crit Care Med
2007
;
176
:
988
93
.
60.
Thomsen
LL
,
Scott
JM
,
Topley
P
,
Knowles
RG
,
Keerie
AJ
,
Frend
AJ
. 
Selective inhibition of inducible nitric oxide synthase inhibits tumor growth in vivo: Studies with 1400W, a novel inhibitor
.
Cancer Res
1997
;
57
:
3300
4
.
61.
Weerakkody
D
,
Moshnikova
A
,
Thakur
MS
,
Moshnikova
V
,
Daniels
J
,
Engelman
DM
, et al
Family of pH (low) insertion peptides for tumor targeting
.
Proc Natl Acad Sci U S A
2013
;
110
:
5834
9
.
62.
Tapmeier
TT
,
Moshnikova
A
,
Beech
J
,
Allen
D
,
Kinchesh
P
,
Smart
S
, et al
The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions
.
Proc Natl Acad Sci U S A
2015
;
112
:
9710
5
.
63.
Roberts
S
,
Strome
A
,
Choi
C
,
Andreou
C
,
Kossatz
S
,
Brand
C
, et al
Acid specific dark quencher QC1 pHLIP for multi-spectral optoacoustic diagnoses of breast cancer
.
Sci Rep
2019
;
9
:
8550
.

Supplementary data