Intranasal Iloprost Prevents Tumors in a Murine Lung Carcinogenesis Model

Chemoprevention of lung cancer in high-risk populations, such as former smokers or survivors of early-stage tobacco-related aerodigestive cancers, is a vital piece of the strategy for reducing lung cancer incidence. Lung cancer interception studies (defined as actively combating carcinogenesis at earlier stages) that relied heavily on epidemiologic data have largely been unsuccessful in clinical trials, while agents that are extensively tested in preclinical models have yielded better results (1, 2). A phase II trial of oral iloprost, a prostacyclin analogue, demonstrated improved endobronchial dysplasia in former smokers (3). Prior preclinical studies predicted efficacy in preventing human lung cancer, as prostacyclin overexpression and iloprost treatment led to reduced tumor number in multiple murine adenocarcinoma models, including cigarette smoke (4–6). These results are promising, however, locally delivered lung cancer chemoprevention agents may be more appealing to high-risk patients, based on perceived effectiveness and tolerability of an inhaled delivery regimen (7). A recently completed phase I trial is investigating inhaled delivery of iloprost for lung cancer prevention in former smokers (NCT02237183). This trial will evaluate the toxicity of inhaled iloprost administered 4 times a day for 2 months. With secondary objectives investigating compliance and iloprost signaling pathway gene expression, this trial will provide valuable data to inform future trials and guide characterization of preclinical models. Since previous mouse chemoprevention models with iloprost intervention used drug delivered orally in chow, it was necessary to confirm similar results with inhaled iloprost to correlate with current human dosing. Agents targeting premalignant lung lesions are more likely to be effective if delivered directly to the airways, as has been previously demonstrated for resveratrol and diindolylmethane (8, 9). An inhaled mouse model of iloprost chemoprevention will also overcome the current lack of availability of oral iloprost for preclinical studies. We investigated intranasal delivery of iloprost in a mouse model of urethane-induced lung adenomas and measured its effect on adenoma number, bronchoalveolar lavage (BAL) macrophages, and iloprost target gene expression.

Thirty-two female FVB/NCrl (FVB) mice (Charles River, Stock #207, 6 weeks of age) were housed in a pathogen-free facility in the Veterinary Care Unit at the Rocky Mountain Regional VA Medical Center (RMRVAMC). Studies were carried out in accordance with the recommendations in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the RMRVAMC Animal Care and Use Committee. Mice were maintained on hardwood bedding with a 12 light/dark cycle; food and water were provided ad libitum. Female FVB mice were used because of the cage aggression observed in male mice with increased handling. After acclimation for 2 weeks, 20 mice were injected intraperitoneally with 100 μL of 1 mg urethane/g body weight dissolved in the 0.9% saline vehicle using a 25 gauge, 5/8 inch needle. Control mice (12) received saline vehicle. Mice were weighed daily for 7 days after urethane injection and weekly for the remainder of the experiment. Nine weeks after urethane exposure, iloprost (Cayman Chemicals) or the saline vehicle (10 urethane exposed mice/group or 6 control mice/group) was administered intranasally at a dose of 5 μg/mouse/100 μL (50 μL per nare with a P200 pipette), 5 days per week for 5 weeks (Fig. 1A). For intranasal delivery, mice were anesthetized with isofluorane, half the iloprost was delivered to one nare, followed by a pause to observe inhalation of iloprost, and then the second half of the dose was delivered to the second nare and observed for inhalation (10, 11). This iloprost treatment regimen was used because previous studies showed that mice tolerate iloprost intranasal treatment for 5 weeks, and small premalignant adenomas are detectable in mice after 6 weeks of urethane exposure and multiplicity starts to plateau at 14 to 16 weeks (11, 12). Following the last iloprost treatment (14 weeks post urethane), mice were sacrificed by a lethal dose of Fatal Plus. Serum and BAL fluid (BALF) were collected as described previously (12). Lesions were dissected from surrounding lung tissue using a dissecting microscope and their diameters measured with digital calipers. Surrounding lung tissue was saved for RNA extraction. BAL cells were harvested by centrifugation, counted, and stained with Wright stain prior to manual differential counting. Statistical analysis using Student t test (for comparisons between 2 groups) or one-way ANOVA with Tukey post-hoc analysis (for comparing more than 2 groups) was conducted using GraphPad Prism (RRID:SCR_002798, version 9.0.2).

Surrounding lung tissue and lesions were collected separately in RNA Later (Qiagen) at the time of harvest. RNA was extracted from these samples using the RNeasy mini kit (Qiagen). mRNA was reverse transcribed using the ABI HC cDNA kit (Thermo Fisher Scientific). qPCR primers (Bio-Rad) for mouse included: Cox2, e-cadherin, carboxylesterase 1 (Ces1), Pparγ, Il1β, and vimentin. qPCR was conducted using standard protocol for Sso Advanced SYBR Green Master Mix (Bio-Rad) on a CFX96 Touch (Bio-Rad). All gene expression data was normalized to the reference gene RPS18 and fold changes were calculated using the 2^−ΔΔC_t method. PCR analysis was conducted in triplicate and statistical analysis was done by t test or ANOVA in GraphPad Prism (RRID:SCR_002798, version 9.0.2).

Lung adenoma number was significantly reduced in iloprost-treated mice compared with their vehicle controls (Fig. 1C); no adenomas were detected in the control mice that did not receive urethane. There was no difference in lesion burden (Fig. 1D) as determined by adenoma diameter. This 14-week model is sufficient to induce adenomas (image of a representative lesion in Fig. 1E), but lesion progression did not advance to adenocarcinoma. BAL macrophage content in adenoma-bearing mice was not increased over that in control mice (not treated with urethane), which is similar to what we observed previously in A/J mice at this early time point, and no difference in BAL macrophage content was detected between the intranasal saline and iloprost treatment groups (Fig. 1F; ref. 13). Iloprost did not negatively affect weight gain in adenoma-bearing mice (Fig. 1B), once again demonstrating its safety as a chemoprevention agent.

RNA was extracted from mouse lung tissues and lung adenomas to measure expression of established iloprost signaling targets (14, 15). In urethane-induced lung adenomas from animals treated with intranasal iloprost compared with saline, Pparγ expression was significantly increased while Cox2, vimentin, and Il1β were significantly decreased (Fig. 2A). E-cadherin and Ces1 had small but insignificant increases with inhaled iloprost exposure (Fig. 2A). In whole lung tissue without lesions, e-cadherin and Pparγ (Fig. 2B and C) had increased expression that approached significance in mice exposed to urethane and inhaled iloprost compared with urethane-only mice (P = 0.06 and 0.08 respectively; Fig. 2D). In whole lung tissues from mice treated with intranasal iloprost, Ces1 was significantly increased in urethane-exposed mice receiving intranasal iloprost compared with urethane alone and approached significance for iloprost alone compared with saline controls (P = 0.07). Il1β exhibited significantly elevated expression in urethane mice compared with controls and treatment with inhaled iloprost returned expression to control levels (Fig. 2G). Vimentin and Cox2 expression were variable within urethane groups and thus significant changes were not observed for inhaled iloprost (Fig. 2E and F). The PCR assays in the current study were limited by wide variability in expression of some genes with exposure to urethane, which could be alleviated by increased number of animals in future studies.

Our study demonstrated that intranasal delivery of iloprost reduces adenoma multiplicity in the urethane murine model of lung premalignant lesions similar to what was demonstrated previously with oral iloprost. Treatment with intranasal iloprost also led to changes in gene expression similar to previous in vivo and in vitro studies with iloprost (5, 14). The urethane model results in 5 to 7 adenomas per animal at 14 to 20 weeks in the FVB strain, which are driven by Q61R Kras (6, 16, 17). To model chemoprevention clinical trials in patients with preexisting precancerous lesions (often found in current and former smokers), we delayed iloprost treatment until after the appearance of premalignant lesions (about 6 weeks) instead of providing it before their appearance as was done in previous studies. We limited iloprost treatment to 5 weeks because the successful intranasal iloprost treatment in a murine model of pulmonary hypertension indicated that mice would tolerate this regimen without toxicity. Future studies should explore the effect of alternate treatment regimens, including delaying iloprost treatment even further to test for efficacy during progression to malignant disease and lengthening the duration of inhaled iloprost exposure. Urethane generates premalignant and malignant lesions in the adenocarcinoma spectrum, and future studies will need to confirm the effect of intranasal iloprost in a model that generates squamous lesions (18) in order to more closely model human trials. This study was limited by investigating a single carcinogen, and the demonstration of inhaled iloprost efficacy would benefit from comparative studies between other single or complex carcinogens, such as cigarette smoke. The intranasal iloprost mouse model is important for its relevance to human disease treatment but is limited by differences in inhaled delivery method. In clinical trials, inhaled iloprost is delivered by the FDA-approved method of nebulization, in which a mist is inhaled into the lungs. The effectiveness of intranasal drug delivery in mice depends on volume of drug delivered and the level of anesthesia (10). While both a nebulizer and intranasal administration deliver medication to the lower respiratory tract, a nebulizer allows for more uniform delivery to distal airways.

Our prior studies showed that oral iloprost treatment prevents roughly 30% of lesions in the urethane model, while intranasal iloprost prevents around 40% (6). Transgenic prostacyclin synthase (PGIStg) expression in mouse lung tissue prevents approximately 60% of lesions, but mice are exposed to increased prostacyclin from birth, compared with 15 weeks of oral iloprost treatment or 5 weeks of intranasal iloprost treatment (6). This study was not powered to directly compare oral and inhaled iloprost, but inhaled iloprost may be a more effective delivery route than oral iloprost, as higher concentrations of drug is delivered to the target organ. Increased effect may also be seen with longer treatment duration, which can be tested in future animal and human studies. Testing the efficacy of intranasal iloprost on regression of more advanced lesions such as adenocarcinoma in situ or later times points using longitudinal monitoring of individual lesions with microCT imaging would yield valuable information. Average BAL macrophage number did not increase with 5 weeks of intranasal iloprost exposure, compared with an increase observed with 15 weeks of oral iloprost (6). It may be that macrophages are part of a systemic response to iloprost in FVB mice, but their numbers have yet to increase at 16 weeks after urethane exposure in FVB mice similar to studies of A/J mice (13). Future studies could explore the efficacy of different iloprost delivery routes and iloprost effects on the infiltration and activity of immune cells in the lungs of premalignant lesion or tumor-bearing mice.

A small number of gene expression changes have been established as measures of prostacyclin or iloprost activity in lung cells. In immortalized human bronchial epithelial cells, PPARγ, CES1, and e-cadherin are reduced by 4 and 8 weeks of exposure to cigarette smoke condensate (CSC) but their expression is increased by the removal of CSC and the addition of iloprost (14). In this same model, COX2 and vimentin are increased with CSC exposure and COX2 is decreased with iloprost treatment (14). In PGIS overexpressing FVB mice, PPARγ and CES1 expression are increased compared to wild type mice (5). In urethane and smoking lung cancer mouse models, e-cadherin, PPARγ, vimentin and COX2 are restored to control levels in PGIS transgenic mice compared with wild-type animals after carcinogen exposure (14). Results from this intranasal iloprost study align with previous studies of iloprost treatment, demonstrating increased epithelial and prostacyclin pathway markers and decreased mesenchymal and inflammatory markers. In this study, PPARγ, vimentin, and COX2 were significantly different in urethane-induced lesions from mice treated with intranasal iloprost compared with saline-treated mice; lesions have not been previously characterized after iloprost treatment, but we expect that lesions arising in PGIStg- or iloprost-treated mice would have reduced expression of the cancer-promoting genes targeted by prostacyclin, similar to whole lung tissue. Numerous potential biomarkers of iloprost exposure and response, including PPARγ and CES1, are being measured in the inhaled iloprost clinical trial, providing a direct comparison with expression of these genes in our animal model. However, differences in the initiating mutations and signaling between adenomas and premalignant bronchial dysplasia may cloud these comparisons. Since iloprost shows efficacy in preventing progression of both mouse adenomas and human bronchial dysplasia, there may be pathways in common between these two diseases, or iloprost may act on stromal cells common to both lesions.

We identified IL-1β as a new marker of iloprost activity, demonstrating increased expression with urethane alone that returned to control levels with intranasal iloprost. IL-1β expression is reduced by half in urethane-induced lesions when mice are treated with iloprost. While IL-1β has not been measured in previous iloprost studies, we do not anticipate that it is unique to the intranasal delivery method. Technical challenges prevented measurement of IL-1β levels in BAL fluid, but future studies will use multiple assays to assess the role of IL-1β in the effects of iloprost. The CANTOS trial found reduced lung cancer risk in patients receiving anti-IL-1β therapy and subsequent studies suggest that the IL-1β inflammatory pathway may contribute to progression of lung cancer (19, 20). However, the nature of the lung cancer risk analysis from the CANTOS trial and the low cost-effectiveness of canakinumab for prevention strategies decrease the appeal of this drug for lung cancer chemoprevention (21–23). Canakinumab is also being studied in multiple lung cancer treatment trials (NCT03447769, NCT03968419, NCT03631199, NCT03626545, NCT03064854). Studies aimed at uncovering iloprost's preventive mechanisms are underway and inhibition of IL-1β by iloprost could be an integral part of reducing protumor inflammatory signaling and intercepting lung tumor development.

Preclinical studies have explored a large range of agents that have chemopreventive efficacy in mice, including some with inhaled administration such as bexarotene, budesonide, picropodophyllin, pioglitazone, and polyphenon E (24–30). Inhaled delivery of bexarotene achieved higher concentrations in the lung compared with systemic administration and reduced toxicity while maintaining its previously demonstrated anticancer activity for adenocarcinoma and squamous cell carcinoma (26). Aerosolized budesonide showed chemopreventive efficacy in early- and late-stage adenoma models, which will support interventions at multiple points in lesion progression (26). Inhaled bexarotene has yet to be tested in humans and inhaled budesonide did not have a significant effect on bronchial dysplasia in a low-dose clinical trial, however, these agents and others represent potential advances in lung cancer chemoprevention. Support for iloprost as a lung cancer chemoprevention strategy is based on extensive preclinical data, a positive clinical trial, minimal side effects, and a reasonable cost profile. Lung cancer chemoprevention research requires mouse models that are relevant to human disease progression and drug delivery approaches. Our study used a chemical carcinogen model that generates lung lesions similar to human disease progression and incorporated a delivery method that is approved for iloprost treatment of pulmonary hypertension. This model of intranasal delivery of iloprost in mice will be critical for preclinical studies investigating mechanisms of iloprost chemoprevention and will support future phase II iloprost chemoprevention trials.

M.A. Tennis reports grants from NIH during the conduct of the study. R.L. Keith reports grants from Department of Veterans Affairs during the conduct of the study; in addition, R.L. Keith has a patent for Uses of Prostacyclin Analogs for Cancer Prevention (62/681,962) issued and with royalties paid from Visiongate. No disclosures were reported by the other authors.

M.A. Tennis: Conceptualization, resources, data curation, supervision, writing–original draft, writing–review and editing. A.J. Smith: Investigation, writing–review and editing. L.D. Dwyer-Nield: Conceptualization, data curation, formal analysis, supervision, investigation, visualization, methodology, writing–original draft, writing–review and editing. R.L. Keith: Conceptualization, resources, supervision, funding acquisition, project administration, writing–review and editing.

This work was supported by the Veterans Administration (Biomedical Laboratory Research and Development Grant Merit Review number BX000382 to R.L. Keith) and NIH grant R01CA214531 to M.A. Tennis. Thank you to Kayla Sompel for preparing the hematoxylin and eosin image in Figure 1. This work was supported by the Veterans Administration (Biomedical Laboratory Research and Development Grant number BX000382).

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