Induced Lung Tumors Research Articles

Short-chain fatty acids induced lung tumor cell death and increased peripheral blood CD4+ T cells in NSCLC and control patients ex vivo.

Despite therapy advances, one of the leading causes of cancer deaths still remains lung cancer. To improve current treatments or prevent non-small cell lung cancer (NSCLC), the role of the nutrition in cancer onset and progression needs to be understood in more detail. While in colorectal cancer, the influence of local microbiota derived SCFAs have been well investigated, the influence of SCFA on lung cancer cells via peripheral blood immune system should be investigated more deeply. In this respect, nutrients absorbed via the gut might affect the tumor microenvironment (TME) and thus play an important role in tumor cell growth. This study focuses on the impact of the short-chain fatty acid (SCFA) Sodium Butyrate (SB), on lung cancer cell survival. We previously described a pro-tumoral role of glucose on A549 lung adenocarcinoma cell line. In this study, we wanted to know if SB would counteract the effect of glucose and thus cultured A549 and H520 in vitro with and without SB in the presence or absence of glucose and investigated how the treatment with SB affects the survival of lung cancer cells and its influence on immune cells fighting against lung cancer. In this study, we performed cell culture experiments with A549, H520 and NSCLC-patient-derived epithelial cells under different SB levels. To investigate the influence on the immune system, we performed in vitro culture of peripheral mononuclear blood cells (PBMC) from control, smoker and lung cancer patients with increasing SB concentrations. To investigate the effect of SB on lung tumor cells, we first analyzed the effect of 6 different concentrations of SB on A549 cells at 48 and 72 hours cell culture. Here we found that, SB treatment reduced lung cancer cell survival in a concentration dependent manner. We next focused our deeper analysis on the two concentrations, which caused the maximal reduction in cell survival. Here, we observed that SB led to cell cycle arrest and induced early apoptosis in A549 lung cancer cells. The expression of cell cycle regulatory proteins and A549 lung cancer stem cell markers (CD90) was induced. Additionally, this study explored the role of interferon-gamma (IFN-γ) and its receptor (IFN-γ-R1) in combination with SB treatment, revealing that, although IFN-γ-R1 expression was increased, IFN-γ did not affect the efficacy of SB in reducing tumor cell viability. Furthermore, we examined the effects of SB on immune cells, specifically CD8+ T cells and natural killer (NK) cells from healthy individuals, smokers, and NSCLC patients. SB treatment resulted in a decreased production of IFN-γ and granzyme B in CD8+ T cells and NK cells. Moreover, SB induced IFN-γ-R1 in NK cells and CD4+ T cells in the absence of glucose both in PBMCs from controls and NSCLC subjects. Overall, this study highlights the potential of SB in inhibiting lung cancer cell growth, triggering apoptosis, inducing cell cycle arrest, and modulating immune responses by activating peripheral blood CD4+ T cells while selectively inducing IFN-γ-R1 in NK cells in peripheral blood and inhibiting peripheral blood CD8+ T cells and NK cells. Thus, understanding the mechanisms of action of SB in the TME and its influence on the immune system provide valuable insights of potentially considering SB as a candidate for adjunctive therapies in NSCLC.

Targeted delivery of doxorubicin by SP5-52 peptide conjugated exosome nanoparticles into lung tumor: An in vitro and in vivo study

Here, SP5-52 peptide conjugated exosome nanostructures were developed for targeted delivery of Doxorubicin (DOX) into lung tumor. Exosomes extracted from the serum of Balb/c mice, were characterized in terms of structure and physicochemical properties. Based on the results, the size and zeta potential of exosome NPs were 67 nm and −5 mV, respectively, which was slightly increased after SP5-52 conjugation and DOX loading. The in vitro cell studies displayed that targeted DOX-loaded exosomes had higher cytotoxicity, cellular uptake, and accumulation in LL/2 cell lines compared with non-targeted DOX-loaded exosomes and control groups. Moreover, the therapeutic efficacy of the prepared formulations was evaluated in lung tumor bearing Balb/c mice within 21 days. Accordingly, targeted DOX-loaded exosomes had effectively treated the lung tumor in comparison to other groups. The rate of survival in the mice treated with targeted exosomes was also 35 % higher than other groups; while, the mortality rate was lower. The histopathological evaluation confirmed the accepted off-targeted properties of the targeted exosomes as lower toxicity was observed in different organs rather than lung. This study presents an effective anticancer drug delivery system for specific targeting of induced lung tumor, which could be useful in the treatment of malignant lung cancers in the future.

14 Carcinogenicity Risk Assessment of Various Carbon Nanotubes by Intra-Tracheal Intra-Pulmonary Spray (TIPS) Dosing Followed by 2-Year Observation

Abstract Multi-walled carbon nanotubes (MWCNTs) are composed of multiple coaxially arranged graphene cylinders. MWCNTs range in diameter from 1 to over 100 nm depending on the number of constituting graphene cylinders. MWCNTs can be divided into two general subtypes, straight and tangled: increasing the number of constituent graphene cylinders increases the number of layers that compose the carbon nanotube wall, and as the number of wall layers increase, the wall thickness increases and the MWCNT becomes more rigid and straight. MWCNTs with a low number of wall layers are flexible and can assemble into tangled agglomerates. We initially showed MWCNT-N, a straight-type MWCNT with 30-40 walls, induced lung tumors and mesotheliomas after administration by intra-tracheal intra-pulmonary spraying (TIPS): rats were administered a total dose of 1mg/rat during the initial 2 weeks of the study, and animals were observed for 2 years without any further treatments. Using this short-term dosing and 2-year observation method we conducted carcinogenicity tests for other MWCNTs with different cylinder wall layers. We found that both straight type MWCNTs, MWCNT-7 (40 layers), MWCNT-A (150 layers), and tangled type MWCNTs, MWCNT-B (15 layers) and double-walled CNT (DWCNT) (2 layers) induced lung tumors and/or pleural mesotheliomas. In contrast to these results, we found carbon nanohorns, carbon nanostructures composed of single-walled carbon nanotubes (SWCNT), and carbon nanobrushes, a fibrous aggregate of carbon nanohorns, were not carcinogenic. Data on foreign body radical production, microarray and mutational signature analyses, and gene alterations will be presented.

Abstract 1479: FOXA2 induces invasive mucinous adenocarcinoma of the lung in the presence of mutant KRAS

Abstract Introduction: The role of the endoderm lineage transcription factor FOXA2 in autochthonous lung tumors has been elusive though the expression of FOXA2 has been reported in human lung cancers. Here, using GEMM (Genetically Engineered Mouse Models) and human lung cancer cell lines, we delineate the role of FOXA2 in lung cancers. Experimental Procedures: GEMM that carry mutant KRAS or mutant EGFR along with FOXA2 (CCSP-rtTA; tetO-Foxa2; tetO-Kras.G12D or tetO-EGFR.L858R) were developed allowing for lung tumor induction by doxycycline administration. The growth of lung tumors affected by FOXA2 was assessed by microCT and pathologically. FOXA2 downstream genes were also evaluated in vivo by immunohistochemistry of mouse lung sections and in vitro by gain (ectopic lentivirus transduction) and loss (siRNA) of function approaches with human lung cancer cell lines using TaqMan gene expression, RNA-seq, ChIP-seq and CRISPRi analyses. FOXA2 protein expression in human lung cancer specimens was also assessed. Data: FOXA2 suppressed the growth of EGFR-mutant lung tumors while promoting that of KRAS-mutant lung tumors in GEMM. Notably, half of the KRAS-mutant lung tumors presented invasive mucinous adenocarcinoma of the lung (IMA) with the expression of a master mucous transcription factor SPDEF and the two major IMA mucin genes MUC5AC and MUC5B. In vitro, FOXA2 induced the expression of SPDEF, MUC5AC and MUC5B by directly binding to their loci. H3K27ac was introduced at the enhancer region of MUC5AC (most highly expressed IMA gene) by FOXA2 and the expression of MUC5AC initiated by FOXA2 was enhanced by mutant KRAS. FOXA2 was highly expressed in human IMA compared to normal lung. Conclusions: FOXA2 induces IMA in the presence of mutant KRAS by directly regulating the expression of mucous genes. Citation Format: Yutaka Maeda, Koichi Tomoshige, William D. Stuart, Iris M. Fink-Baldauf, Masaoki Ito, Tomoshi Tsuchiya, Takeshi Nagayasu, Tomoki Yamatsuji, Morihito Okada, Takuya Fukazawa, Minzhe Guo. FOXA2 induces invasive mucinous adenocarcinoma of the lung in the presence of mutant KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1479.

Abstract 1797: Impact of estrogen depletion using an aromatase inhibitor (AI) on EGFR-mutated non-small cell lung cancer (NSCLC) in a murine model

Abstract EGFR-mutated lung tumors respond poorly to checkpoint (PD-1/PD-L1) inhibitors. Resistance to such therapies has been attributed to the prototypic immunosuppressed “cold” tumor microenvironment (TME) of EGFR-mutated NSCLCs, which is characterized by low levels of tumor infiltrating lymphocytes. Growing evidence from breast cancer studies demonstrates the ability of 17β-estradiol to regulate several species of immune cells within an immunosuppressive TME. Furthermore, treatment of breast cancers with AIs or estrogen receptor antagonists promotes lymphocyte infiltration and enhances immune reactivity. While the role of estrogen species in lung tumorigenesis is well documented, little attention has been given to their contribution to the immunosuppressed lung TME or the use of estrogen-modulating agents to attenuate these effects. The goal of this study was to evaluate the impact of estrogen depletion, using the AI exemestane (Exe), on levels of tumor infiltrating immune cells and other tumor endpoints in the murine model of mutated EGFRL858R NSCLC. Male (n = 42) and female (n = 39) mutEGFRL858R mice were gonadectomized at 6 weeks of age and doxycycline-supplemented chow was given to induce lung tumor development. Daily treatment with Exe (250 μg/kg) or vehicle was initiated at 8 weeks of age, and continued for the duration of the experiment (7 weeks). Body weights were recorded twice per week. At the time of euthanasia, lung tissues were processed for histopathological analysis and assessment of tumor CD8+ lymphocyte concentrations by immunohistochemistry was performed. Lung tumors from Exe-treated mice exhibited higher concentrations (median, Q1 - Q3) of CD8+ T cells per 20x field (77.5, 60.3 - 114.0) vs. those of vehicle-treated controls (61.0, 48.8 - 80.8) (P = 0.035). A trend of a decreased incidence of solid adenocarcinoma lesions within the lungs of Exe-treated vs. vehicle-treated control mice was observed in both males (26.3% vs. 38.1%) and females (26.3% vs. 35.0%). Among females, the incidence of continuous weight loss (over 3 consecutive weeks) was lower among Exe-treated (16.7%) vs. vehicle-treated (40.0%) mice. Early findings suggest that administration of AI therapy to mutEGFRL858R mice stimulates CD8+ lymphocyte infiltration within lung tumors, and inhibits both the formation of lung adenocarcinomas and disease-associated weight loss. Murine studies to further optimize the dose and schedule of AI treatment, as well as evaluate the ability of AI therapy to enhance the activity of checkpoint inhibitors in mutated EGFRL858R NSCLC are underway. (Supported by an In Vino Vita Award from Fox Chase Cancer Center and a R01 CA217161 from the NCI/NIH.) Citation Format: J. Nicholas Bodor, Rodrigo T. Macedo, Kristen Harvey, Lisa A. Vanderveer, Daniel D. Krzizike, Martin J. Edelman, Joseph Treat, Margie L. Clapper. Impact of estrogen depletion using an aromatase inhibitor (AI) on EGFR-mutated non-small cell lung cancer (NSCLC) in a murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1797.

MRNA expression levels of CCL4, IL6, and CXCL2 in multiwalled carbon nanotube induced lung tumors in rats

mRNA expression levels of CCL4, IL6, and CXCL2 in multiwalled carbon nanotube induced lung tumors in rats

Comparative pulmonary and genotoxic responses to inhaled nickel subsulfide and nickel sulfate in F344 rats.

Inhalation studies with nickel (Ni) subsulfide (Ni3 S2 ) and Ni sulfate hexahydrate (NiSO4 ·6H2 O) investigated differences in mode of action that could explain why the former induced lung tumors in rats and the latter did not. Male rats were exposed to ≤0.22 mg Ni/m3 NiSO4 ·6H2 O or 0.44 mg Ni/m3 Ni3 S2 , 6 h/day, 5 days/week for 3 and 13 weeks; subsets of the rats exposed for 13 weeks were held for an additional 13 weeks. Analyses of bronchoalveolar lavage fluid, isolated cells, and whole lung tissue were conducted to compare the extent and persistence of any induced lung effects. Histological findings were qualitatively identical for both compounds and consistent with lesions reported in earlier studies. After 13 weeks of exposure, the incidence and severity of pulmonary inflammation and epithelial cell hyperplasia were greater among Ni3 S2 -exposed rats, whereas the reverse response was seen for apoptosis. Only Ni3 S2 exposure significantly increased epithelial and non-epithelial cell proliferation after 13 weeks of exposure. Both compounds induced DNA damage in isolated lung cells and DNA hypermethylation of whole lung tissue after 13 weeks of exposure at the highest exposure concentrations. Increases in cell proliferation, DNA damage, and tissue DNA hypermethylation did not persist during the 13-week recovery period. In summary, the highest concentrations of each compound produced marked pulmonary toxicity, but the lowest concentrations produced minimal or no effects. Differences in the proliferative and apoptotic responses between the two compounds may help explain differences in carcinogenicity, whereas the identification of no observed adverse effect concentrations (NOAECs) contributes to the risk characterization for inhalation exposure to nickel compounds.

Monitoring autochthonous lung tumors induced by somatic CRISPR gene editing in mice using a secreted luciferase

BackgroundIn vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies.MethodsTo facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes.ResultsWe demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination.ConclusionsOur study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.

Two-year intermittent exposure of a multiwalled carbon nanotube by intratracheal instillation induces lung tumors and pleural mesotheliomas in F344 rats

BackgroundA mounting number of studies have been documenting the carcinogenic potential of multiwalled carbon nanotubes (MWCNTs); however, only a few studies have evaluated the pulmonary carcinogenicity of MWCNTs in vivo. A 2-year inhalation study demonstrated that MWNT-7, a widely used MWCNT, was a pulmonary carcinogen in rats. In another 2-year study, rats administered MWNT-7 by intratracheal instillation at the beginning of the experimental period developed pleural mesotheliomas but not lung tumors. To obtain data more comparable with rats exposed to MWNT-7 by inhalation, we administered MWNT-7 to F344 rats by intratracheal instillation once every 4-weeks over the course of 2 years at 0, 0.125, and 0.5 mg/kg body weight, allowing lung burdens of MWNT-7 to increase over the entire experimental period, similar to the inhalation study.ResultsAbsolute and relative lung weights were significantly elevated in both MWNT-7-treated groups. Dose- and time-dependent toxic effects in the lung and pleura, such as inflammatory, fibrotic, and hyperplastic lesions, were found in both treated groups. The incidences of lung carcinomas, lung adenomas, and pleural mesotheliomas were significantly increased in the high-dose group compared with the control group. The pleural mesotheliomas developed mainly at the mediastinum. No MWNT-7-related neoplastic lesions were noted in the other organs. Cytological and biochemical parameters of the bronchoalveolar lavage fluid (BALF) were elevated in both treated groups. The lung burden of MWNT-7 was dose- and time-dependent, and at the terminal necropsy, the average value was 0.9 and 3.6 mg/lung in the low-dose and high-dose groups, respectively. The number of fibers in the pleural cavity was also dose- and time-dependent.ConclusionsRepeated administration of MWNT-7 by intratracheal instillation over the 2 years indicates that MWNT-7 is carcinogenic to both the lung and pleura of rats, which differs from the results of the 2 carcinogenicity tests by inhalation or intratracheal instillation.

Mechanistic insight into the synergism of IL-27 and IL-28B in regulation of benzo(a)pyrene-induced lung carcinogenesis associated ROS/NF-κB/NLRP3 crosstalk

AimOur previous work depicted that benzo(a)pyrene (BaP)-induced lung cancer associated pulmonary redox imbalance and inflammation were effectively regulated by the combinatorial treatment of IL-27 and IL-28B. So in continuation of that finding the present study was designed to reveal the inflammation regulating signaling network modulated by IL-27 and IL-28B treatment related to BaP-induced lung cancer. MethodsMale Swiss albino mice were treated with BaP to induce lung tumor. Then they received individual as well as combinatorial treatment of IL-27 and IL-28B. At the end of the experimental schedule, the expression of NF-κB signaling proteins, the formation of NLRP3 inflammasome complex and IL-18; IL-17A expression in the lung were observed using Western blot and RT-PCR. The tissue and serum levels of some proinflammatory cytokines were also studied using ELISA. Mast cell density was also studied using toluidine blue staining procedure. ResultsTreatment with IL-27 or IL-28B alone was successful to regulate the expression of NF-κB signaling proteins and NLRP3 complex in some cases but best attenuation was observed in animals who received both IL-27 and IL-28B in combination. In combination, it was successful in down-regulating the expression of p-ERK1/2 and in reducing the accumulation of mast cells in the lung tissue associated with BaP-induced lung carcinogenesis. The impaired PPARγ expression was also reinstated upon combination treatment. ConclusionAltogether, the treatment in combination with IL-27 and IL-28B is an effective regimen to attenuate the ROS/NF-κB/NLRP3 axis associated with BaP-induced lung carcinogenesis.

303 Non-functional T cell responses in non-small cell lung cancer are induced during tumor antigen-specific T cell priming in the mediastinal lymph node

BackgroundIn non-small cell lung cancer (NSCLC), response to checkpoint blockade therapy (CBT) is associated with tumor-infiltrating CD8+ T cells, but not all T cell-infiltrated tumors respond to CBT. The subgroup of T cell-infiltrated but CBT-resistant tumors has been clinically described as containing ”non-functional” T cell responses. Mechanisms governing the generation of non-functional T cell responses remain poorly understood, and treatment options for this subgroup are limited.MethodsWe utilized a transplantable, syngeneic murine NSCLC cell line derived from an autochthonous NSCLC driven by KrasG12D expression and p53 deletion (KP cell line) to model non-functional T cell responses. To study antigen-specific responses, we engineered KP cells to express the model CD8+ T cell antigen SIY for certain experiments. CBT consisted of combined anti-CTLA-4 and anti-PD-L1 therapy.ResultsOrthotopic KP lung tumors failed to respond to CBT, but KP flank tumors were controlled by CBT. Lung and flank tumors contained activated CD8+ T cells, providing a platform to compare functional and non-functional CD8+ T cell responses in NSCLC. Single-cell RNA sequencing revealed that lung tumor-infiltrating CD8+ T cells lacked effector and exhaustion molecules despite clonal expansion. Analysis of antigen-specific CD8+ T cells revealed that this lung cancer-specific T cell dysfunction was established during priming in lung-draining mediastinal lymph nodes (mLN) despite robust T cell proliferation. RNA sequencing and flow cytometry of antigen-specific CD8+ T cells found that T cells primed in the mLN underwent blunted effector differentiation characterized by a lack of effector molecules CD25, Granzyme B, and TIM-3, but retention of TCF-1. This phenotype persisted in lung tumors, consistent with our initial observations of absent effector and exhaustion molecule expression. Many CD8+ T cells from NSCLC patients expressed an analogous gene expression program distinct from T cell exhaustion. TCF-1+ CD8+ T cells in lung tumors did not mediate tumor control and failed to differentiate into effector cells after CBT. To investigate alternative therapeutic strategies of reinvigorating lung tumor-reactive T cells, we focused on IL-2 and IL-12, as expression of their receptors was reduced in mLN-primed T cells. Administering recombinant IL-2 and IL-12 was sufficient to restore effector T cell differentiation, induce lung tumor control, and significantly extend survival of lung tumor-bearing mice.ConclusionsOur results suggest that non-functional CD8+ T cell responses in NSCLC arise from failed effector T cell differentiation during priming. Transient combination therapy with IL-2 and IL-12 overcomes this dysfunctional state to induce protective T cell responses in CBT-resistant tumors.Ethics ApprovalAll mouse experiments were approved by MIT’s Committee on Animal Care (CAC) - DHHS Animal Welfare Assurance # D16-00078

KRAS4A induces metastatic lung adenocarcinomas in vivo in the absence of the KRAS4B isoform

In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a KrasFSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras+/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras+/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences.

The effect of constant light and melatonin on urethane-induced lung carcinogenesis in mice and expression of clock genes and proteins

Introduction. Circadian rhythms are an adaptational mechanism to day-night cycle. At the cellular level rhythms are supported by oscillations of transcription of Bmal1, 2, Clock and Npas2, Per1-3, Cry1, 2 clock genes; in the organism rhythms are synchronized with melatonin, the "hormone of darkness". Reliable data have been obtained that disruption of biological "clocks" at the cellular and/or organism level are associated with carcinogenesis, but the experimental results in this direction remain incomplete.
 Aim. Assessment of the continuous lighting and melatonin administration effects on chemically induced carcinogenesis in mice and expression of clock genes and proteins in normal and tumor lung tissue.
 Materials and methods. Lung tumors were induced by urethane in 120 male SHR mice. Animals were kept at constant (LL) or standard (LD) light; half of the animals received melatonin (MT) daily at 20 mg/l at night with water for 45 weeks of experiment. At the end of the experiment, the frequency and multiplicity of lung tumours of different size were assessed. Expression of clock genes Clock, Bmal1, Cry1 was assessed in tumor samples and normal lung tissue by real-time PCR; protein content of BMAL1, CLOCK, CRY1 and PER2 was determined by immunohistochemistry. Statistical data were processed using the programs MS Excel 2007, GraphPad Prism 6.0 with commonly used statistical criteria.
 Results. The number of animals with lung tumors was between 89 and 100% in different groups. Large tumors (>2 mm) were more frequently observed in LL group (62 tumors out of 294, 21,1%) than in LD group (59 tumors out of 405, 14,6%, p=0,0245). Administration of exogenous melatonin at constant lighting statistically significantly reduced the frequency of large tumors (28 tumors out of 320, 8.8%, p=0,0001 in comparison with LL). In normal lung tissue of animals kept at constant lighting (LL) the increase in relative expression of clock genes in comparison with LD group was revealed: Bmal1 by 3,1 times (p=0,02) and Cry1 by 3,6 times (p=0,0002). No such differences were found for Clock gene. The relative content of BMAL1 and CLOCK proteins in all experimental conditions was higher in adenomas and adenocarcinomas compared to normal tissue.
 Conclusion. Constant lighting promotes the development of chemically induced lung tumors, and melatonin administration inhibits carcinogenesis under constant lighting.
 The content of BMAL1 and CLOCK transcription activator proteins in lung tumors was found to increase in comparison with normal tissue, whereas no increase in the expression level of corresponding genes in tumors was observed.

Coexposure to Inhaled Aldehydes or Carbon Dioxide Enhances the Carcinogenic Properties of the Tobacco-Specific Nitrosamine 4-Methylnitrosamino-1-(3-pyridyl)-1-butanone in the A/J Mouse Lung.

Tobacco smoke is a complex mixture of chemicals, many of which are toxic and carcinogenic. Hazard assessments of tobacco smoke exposure have predominantly focused on either single chemical exposures or the more complex mixtures of tobacco smoke or its fractions. There are fewer studies exploring interactions between specific tobacco smoke chemicals. Aldehydes such as formaldehyde and acetaldehyde were hypothesized to enhance the carcinogenic properties of the human carcinogen, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) through a variety of mechanisms. This hypothesis was tested in the established NNK-induced A/J mouse lung tumor model. A/J mice were exposed to NNK (intraperitoneal injection, 0, 2.5, or 7.5 μmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 17 ppmv) for 3 h in a nose-only inhalation chamber, and lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had more adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase the severity of NNK-induced lung adenomas. The aldehyde coexposure did not affect the levels of NNK-derived DNA adduct levels. Similar studies tested the ability of a 3 h nose-only carbon dioxide (0, 5, 10, or 15%) coexposure to influence lung adenoma formation by NNK. While carbon dioxide alone was not carcinogenic, it significantly increased the number of NNK-derived lung adenomas without affecting NNK-derived DNA damage. These studies indicate that the chemicals in tobacco smoke work together to form a potent lung carcinogenic mixture.

Overexpression of PRDX4 Modulates Tumor Microenvironment and Promotes Urethane-Induced Lung Tumorigenesis.

Peroxiredoxin 4 (PRDX4), initially reported as an antioxidant, is overexpressed in lung cancer and participates in its progression. However, its role in the urethane-induced lung tumor model is undetermined. The aim of this study was to investigate the effect of PRDX4 overexpression on carcinogen-induced lung tumor development. Human PRDX4 overexpression transgenic (Tg) mice (hPRDX4+/+) and non-Tg mice were intraperitoneally injected with urethane to induce lung tumor. After 6 months, tumor formation was compared between groups and possible mechanisms for the difference in tumor development were investigated. The serum and lung PRDX4 expressions were enhanced after urethane stimulation in Tg mice. Both the average number of tumors (≥0.5 mm) and tumor diameter per mouse in the Tg group were significantly larger than in non-Tg controls, while body weight was lower in the Tg group. Compared with non-Tg controls, tumor cell proliferation was enhanced, while tumor cell apoptosis was suppressed in Tg mice. Systemic oxidative stress and oxidative stress in lung tumors were inhibited by PRDX4 overexpression. The balance of prooxidant enzymes and antioxidant enzymes was also shifted to a decreased level in Tg tumor. In lung tumor tissue, the density of microvessel penetrated into tumor was higher in the Tg group; macrophage infiltration was enhanced in Tg tumors, while there was no difference in T lymphocyte infiltration; the expressions of cytokines, including interleukin-1 beta (IL-1β) and matrix metallopeptidase 9 (MMP9), were elevated in Tg tumors, which resulted from enhanced phosphorylation of nuclear factor-κB p65 (NF-κB p65) and c-Jun, respectively. In conclusion, PRDX4 overexpression modulated tumor microenvironment and promoted tumor development in the mouse urethane-induced lung cancer model.

Abstract 6282: Olaparib in combination with hemithoracic radiation increases the therapeutic index in a urethane-induced mouse lung cancer model

Abstract Purpose: The radiosensitizing effect of the Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib on tumors has been reported in preclinical models. However, its effect on normal tissue toxicity following thoracic radiation have not been well studied. Herein, we investigated the therapeutic index of olaparib combined with hemithoracic radiation after early or late intervention in a urethane-induced lung cancer model. Methods and Materials: Female A/J mice were treated with 5% urethane (i.p.) to induce lung tumors. To assess tolerability, mice first received 13 Gy whole thorax radiation in combination with olaparib (100 mg/kg, p.o.) or vehicle, 1 h before and 2 h after radiation. Body weight was monitored and esophagi were collected 2 wks after treatment. In the early intervention study, treatment started at 8 weeks after urethane injection, mice were treated with olaparib (50 mg/kg, p .o.) or hemithorax irradiation (5 × 5 Gy on left lung) alone and in combination. Mice were sacrificed at 24 wks after radiation treatment. Lung fibrosis was assessed by Masson's Trichrome staining and α-SMA IHC. In the late intervention study, treatment started at 18 weeks after urethane injection. Mice were treated with olaparib (100 mg/kg, p.o.) or hemithorax irradiation (13 Gy on left lung) alone and in combination. Lung tumor growth was monitored by MRI. Mice were sacrificed at 6 wks after radiation treatment. Superficial lung tumors were counted and lung sections were processed for histology. Results: Enhanced body weight loss and evidence of esophageal toxicity were observed when olaparib was combined with whole thorax but not with hemithorax radiation. In the early intervention study, olaparib did not enhance radiation-induced lung fibrosis. However, olaparib plus hemithorax radiation significantly reduced the tumor burden in left lungs compared to radiation alone (total tumor number: p<0.05, tumor volume: p<0.05, surface tumor number: p<0.0001). Similar to the early intervention study, late intervention with olaparib and hemithorax radiation was well-tolerated and also significantly decreased tumor burden compared to hemithorax radiation treatment alone (p<0.05). Conclusions: Our data demonstrate that combination treatment with olaparib and can increase the effectiveness of hemithorax radiation in a urethane-induced lung cancer model in both early and late intervention settings without increasing normal tissue toxicity. Our study provides preclinical support for the ongoing clinical trials of PARP inhibitors in combination with thoracic radiation for lung cancer. Citation Format: Yanyan Jiang, Anderson Ryan. Olaparib in combination with hemithoracic radiation increases the therapeutic index in a urethane-induced mouse lung cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6282.

Abstract A19: Interactions between tobacco smoke chemicals in rodent tumor models

Abstract Tobacco is a complex chemical mixture, containing many toxicants and carcinogens. Most rodent risk assessment studies have focused on single chemicals or the complicated mixtures of tobacco smoke or its fractions. There are few studies evaluating how specific chemicals interact with one another to form the potent carcinogenic mixture of tobacco smoke. We hypothesized that tobacco smoke aldehydes like formaldehyde and acetaldehyde could enhance the carcinogenic properties of the tobacco-specific nitrosamines, N’-nitrosonornicotine (NNN) and 4-methylnitrosamine-1-(3-pyridyl)-1-butanone (NNK), through a variety of mechanisms. This hypothesis was tested in two established rodent tumor models, the NNN-induced rat esophageal tumor model and the NNK-induced A/J mouse lung tumor model. In the first model, rats were exposed to 0, 4, or 8 ppm NNN in the drinking water in the presence or absence of 3000 ppm acetaldehyde for up to 100 weeks. The number of esophageal papillomas per rat was doubled in animals receiving both acetaldehyde and 8 ppm NNN (0.5 versus 1.15 tumors/rat, respectively). Acetaldehyde alone did not cause esophageal tumors. DNA adduct levels were not affected by the combination of the two chemicals. In the second model, A/J mice were exposed to NNK (i.p, 0, 2.5, or 7.5 μmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 15 ppmv) for 3 hours in a nose-only inhalation chamber. Lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had an increased number of adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase dysplasia in tumors initiated by NNK. As in the rat study, DNA adduct levels were not affected by the coexposure. In a separate experiment, we tested the hypothesis that the elevated levels of carbon dioxide in tobacco smoke could affect the carcinogenic properties of NNK in the A/J mouse; tobacco smoke contains 12.5% carbon dioxide. Mice received a 3 h nose-only carbon dioxide (0, 5, 10, or 15%) coexposure of mice receiving NNK (i.p. in saline, 0, 2.5, or 7.5 μmol). The coexposure to carbon dioxide more than doubled the number of lung adenomas induced by 2.5 μmol NNK, with the maximal effect observed with 10% carbon dioxide (0%: 1.8 ± 1.9; 5%: 3.9 ± 2.8; 10%: 7.1 ± 3.5; 15%; 5.5 ± 2.9 lung adenomas/mouse). Lung adenomas were also significantly increased in mice receiving 7.5 μmol NNK, although to a lesser extent (0%: 11 ± 6.2; 5%: 13 ± 7.9; 10%: 18 ± 6.0; 15%; 12 ± 5.3 lung adenomas/mouse). This additive and synergistic effect of carbon dioxide was highly significant (p value = 1.0 × 10-14). The mechanism of this interaction is under investigation. Collectively, these studies support the hypothesis that the aldehydes and carbon dioxide present in the tobacco mixture interact to enhance the carcinogenic potency of the tobacco specific nitrosamines. (Funded by CA-184987.) Citation Format: Lisa A. Peterson, Marissa K. Oram, Donna E. Seabloom, William E. Smith, Alessia Stornetta, Karin R. Vevang, Monica Flavin, Alexandru F. Tabaran, Ingrid Cornax, M. Gerard O’Sullivan, Pramod Upadhyaya, Lin Zhang, Stephen S. Hecht, Silvia Balbo, Timothy S. Wiedmann. Interactions between tobacco smoke chemicals in rodent tumor models [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A19.

Abstract A53: Demonstration of the carcinogenicity of a flexible tangled multiwalled carbon nanotube in the rat lung

Abstract Carbon nanotubes (CNTs) are composed of one or more one-atom thick graphene cylinders. CNTs have remarkable mechanical, thermal, chemical, and electrical properties, and their tubular structure makes them very lightweight. However, these properties also make CNTs easily airborne and highly biopersistent, and respired CNTs have the potential to induce inflammatory lesions in tissues. Similar to asbestos, several MWCNTs were shown to induce peritoneal malignant mesotheliomas after intraperitoneal administration in rats and mice. MWCNT-N was the first CNT shown to induce lesions in lung and pleural tissue after administration via the airway: in this study, MWCNT-N was administered to rats using intratracheal, intrapulmonary spraying (TIPS). Shortly thereafter, MWCNT-7 was shown to induce bronchioloalveolar tumors in rats after inhalation exposure. These two MWCNTs have walls of more than 40 layers and form rod-shaped aggregates, and consequently have the potential to act similarly to asbestos and induce neoplastic lesions in the lung and/or pleural tissue. To investigate the effects of wall thickness on the carcinogenicity of MWCNTs, we conducted a 2-year study comparing 2 different types of MWCNTs, rigid rod-shaped MWCNT-A (213 layers, 150 nm in diameter, forming rigid rod shape aggregates) and flexible MWCNT-B (15-18 layers, 15 nm in diameter, forming tangled shaped aggregates). Crocidolite asbestos was used as the reference material. The rats were administered MWCNTs and asbestos using TIPS once a week over an 8-week period (total low and high doses of 0.5 and 1.0 mg/rat), followed by 2-year observation without further treatment (Suzui et al., 2016; Xu et al., 2014; Abdelgied and Elgazzar, 2019). In the MWCNT-B group, the combined incidence of bronchioloalveolar tumors was increased in the group administered 1.0 mg MWCNT-B compared to the vehicle control group. Malignant pleural mesothelioma was not induced in any of the groups. MWCNT-A and crocidolite asbestos also induced bronchioloalveolar tumors; however, the incidence was not statistically significant compared to the controls. The results indicate that the carcinogenic potential of inhaled flexible, tangled MWCNT-B is as high or higher than that of rigid, rod-shaped MWCNT-A. This is the first study to show that a thin, flexible MWCNT induces lung tumors after administration via the airway. Further studies are warranted to investigate the association of shape and rigidity with the carcinogenicity of respirable CNTs. Citation Format: Hiroyuki Tsuda, William T. Alexander, Takamasa Numano, Mohamed Ahmed Mahmoud Abdelgied, Ahmed Maher Mahmoud Ibrahim Elgazzar, Hiroshi Takase, Jiegou Xu, Aya Naiki, Satoru Takahashi, Akihiko Hirose, Makoto Ohnishi, Jun Kanno, David B. Alexander. Demonstration of the carcinogenicity of a flexible tangled multiwalled carbon nanotube in the rat lung [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A53.

Modulation of the PD-1/PD-L1 immune checkpoint axis during inflammation-associated lung tumorigenesis.

Chronic obstructive pulmonary disease (COPD) is a significant risk factor for lung cancer. One potential mechanism through which COPD contributes to lung cancer development could be through generation of an immunosuppressive microenvironment that allows tumor formation and progression. In this study, we compared the status of immune cells and immune checkpoint proteins in lung tumors induced by the tobacco smoke carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or NNK + lipopolysaccharide (LPS), a model for COPD-associated lung tumors. Compared with NNK-induced lung tumors, NNK+LPS-induced lung tumors exhibited an immunosuppressive microenvironment characterized by higher relative abundances of PD-1+ tumor-associated macrophages, PD-L1+ tumor cells, PD-1+ CD4 and CD8 T lymphocytes and FOXP3+ CD4 and CD8 T lymphocytes. Also, these markers were more abundant in the tumor tissue than in the surrounding 'normal' lung tissue of NNK+LPS-induced lung tumors. PD-L1 expression in lung tumors was associated with IFNγ/STAT1/STAT3 signaling axis. In cell line models, PD-L1 expression was found to be significantly enhanced in phorbol-12-myristate 13-acetate activated THP-1 human monocytes (macrophages) treated with LPS or incubated in conditioned media (CM) generated by non-small cell lung cancer (NSCLC) cells. Similarly, when NSCLC cells were incubated in CM generated by activated THP-1 cells, PD-L1 expression was upregulated in EGFR- and ERK-dependent manner. Overall, our observations indicate that COPD-like chronic inflammation creates a favorable immunosuppressive microenvironment for tumor development and COPD-associated lung tumors might show a better response to immune checkpoint therapies.

Mechanism of Action of TiO2: Recommendations to Reduce Uncertainties Related to Carcinogenic Potential.

The Risk Assessment Committee of the European Chemicals Agency issued an opinion on classifying titanium dioxide (TiO2) as a suspected human carcinogen upon inhalation. Recent animal studies indicate that TiO2 may be carcinogenic through the oral route. There is considerable uncertainty on the carcinogenicity of TiO2, which may be decreased if its mechanism of action becomes clearer. Here we consider adverse outcome pathways and present the available information on each of the key events (KEs). Inhalation exposure to TiO2 can induce lung tumors in rats via a mechanism that is also applicable to other poorly soluble, low-toxicity particles. To reduce uncertainties regarding human relevance, we recommend gathering information on earlier KEs such as oxidative stress in humans. For oral exposure, insufficient information is available to conclude whether TiO2 can induce intestinal tumors. An oral carcinogenicity study with well-characterized (food-grade) TiO2 is needed, including an assessment of toxicokinetics and early KEs.