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BACKGROUND: Secondhand smoke (SHS) contains more than 7000 chemicals, 70of which are carcinogens and it is estimated that every one in four Americans areinvoluntarily exposed to SHS. Carcinogenesis induced by SHS exposure isunclear, though SHS exposure is associated with various cancers including headand neck squamous cell carcinoma (HNSCC). Recently, it has been shown thatSHS exposure is a significant independent predictor of HNSCC recurrence. Wehave shown that in vitro exposure to SHS increases HNSCC cisplatin resistance. AIMS: Our study examines the effect of SHS smoke exposure on cisplatin-induced HNSCC cancer cell death and the possible mechanisms behind thisobservation. METHODS: Sidestream smoke (SS), the main component of SHS,was extracted as previously described. Authenticated HNSCC cell lines (UM-SCC1 & WSU-HN6) were exposed to SS extract for 48 hours at doses mimickingthe nicotine levels observed in the saliva of passive smokers. Then, cancer cellswere treated with cisplatin in the presence or absence of SS extract. Cisplatin-induced cancer cell death was measured using Incucyte® S3 Live-Cell imaginganalysis. Transcriptomic analysis following RNA-sequencing was performedusing Ingenuity Pathway Analysis. Data were subjected to ANOVA. RESULTS: Our live-cell imaging system revealed that HNSCC cells exposed to SS extractsignificantly decreased cisplatin-induced cancer cell death at various time points compared with cells treated with cisplatin alone in both cell lines. The HNSCCcells treated only with SS extract did not show any change in cell death at 0, 12,24, 48, 72, and 96 hours. RNA-sequencing data revealed that cell death andsurvival pathways are the most changed pathways in the SS extract-treatedgroup compared with unexposed HNSCC cells. CONCLUSIONS: Our studydocuments for the first time that even short-term exposure to SHS allows cells toevade cisplatin-induced cell death and can lead to head and neck cancercisplatin resistance. At the molecular level, SHS exposure alters genes that areassociated with cell death and survival. Further, HNSCC patients-basedobservations and mechanistic studies are warranted. FUNDING: This work was partially supported by the National Cancer Institute of the NationalInstitutes of Health (R33CA202898, R01CA242168, and P30CA225520), theTSET Health Promotion Cancer Center, and the Oklahoma Center forAdvancement of Science and Technology (HR16-007). Dr. Queimado holds aPresbyterian Health Foundation Endowed Chair in Otorhinolaryngology Position. Citation Format: Balaji Sadhasivam, Jimmy Manyanga, Vengatesh Ganapathy, Célia Bouharati, David Rubenstein, Jonathan Wren, Pawan Acharya, Daniel Zhao, Lurdes Queimado. Secondhand smoke exposure alters cisplatin-induced cell death and alters cell death and survival pathway in HNSCC [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 384.

Background: E-cigarette use has skyrocketed among youth, in part because e-cigarettes are perceived as a safer substitute for traditional cigarettes. However, the resulting health effects of e-cigarette use are still unclear. E-cigarette aerosols contain harmful and potentially harmful substances such as flavorings, carbonyl compounds, heavy metals, carcinogens and reactive oxygen species (ROS). A recent in vitro study from our laboratory showed that e-cigarette aerosols can increase cellular ROS and suppress cellular antioxidant capacity. An imbalance between the ROS production and the availability of antioxidants or free radical scavengers, can lead to chronic inflammation. Here we examine the effect of exposure to e-cigarette aerosols on the expression of the inflammatory regulator NF-kB and its downstream inflammatory targets in oral epithelial cells. Methods: Human oral epithelial cancer (UM-SCC-1) cells were exposed, every other day for 2 weeks, to e-cigarette aerosol extracts (18 mg/ml of nicotine; tobacco flavor) prepared from two distinct e-cigarette brands. Standard tobacco extracts were used as a positive control. Whole-cell RNA was isolated and processed for RNA-sequencing. The altered gene expression was further validated by western blotting and ELISA using a human cytokine array. Data were analyzed by Student's t-test. Results: RNA sequencing data showed that a 2-week exposure of oral epithelial cells to e-cigarette aerosol extracts resulted in significant changes in several key cellular signaling pathways, including inflammatory and immune response signals. Exposure to e-cigarette aerosol extracts resulted in a significant increase in the expression of proteins involved in inflammatory pathways, such as TLR3, TGF beta, ERK1/2 and NF-kB. We also observed a significant increase in pro-inflammatory and anti-inflammatory cytokines after exposure to e-cigarette aerosol extracts, including CD54, IL-1a and IL-10. Conclusion: Our data suggest that chronic exposure to e-cigarette aerosol increases the expression of TLR3, TGF beta, and ERK1/2, which possibly contributed to the observed increase in NF-kB and its down-stream targets (e.g., CD54, IL-1a and IL-10). NF-kB is a pleiotropic transcription factor with key roles in multiple biological processes, such as immune homeostasis and development. Chronic NF-kB activation by e-cigarette aerosol exposure could contribute to chronic inflammation and tumorigenesis. Citation Format: Vengatesh Ganapathy, Jimmy Manyanga, Dehra McGuire, Daniel Brobst, Balaji Sadhasivam, Mayilvanan Chinnaiyan, Constantin Georgescu, Jonathan Wren, Lurdes Queimado. E-cigarette aerosol exposure increases NF-kB and modulates inflammatory markers in oral epithelial cells. [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 4470.

Tobacco smoking is the leading preventable cause of cancer. Moreover, continued smoking during cancer therapy reduces overall survival. Aware of the negative consequences of tobacco smoking and the challenges of smoking cessation, cancer patients are inquiring whether they should switch to electronic cigarettes (e-cigarettes). To obtain evidence-based data to inform this decision, we examined the effects of e-cigarette aerosol exposure on cisplatin resistance in head and neck cancer cells. Our results show that cancer cells exposed to e-cigarette aerosol extracts and treated with cisplatin have a significant decrease in cell death, increase in viability, and increase in clonogenic survival when compared to non-exposed cells. Moreover, exposure to e-cigarette aerosol extracts increased the concentration of cisplatin needed to induce a 50% reduction in cell growth (IC50) in a nicotine-independent manner. Tobacco smoke extracts induced similar increases in cisplatin resistance. Changes in the expression of drug influx and efflux transporters, rather than activation of cell growth-promoting pathways or DNA damage repair, contribute to e-cigarette induced cisplatin resistance. These results suggest that like combustible tobacco, e-cigarette use might increase chemotherapy resistance, and emphasize the urgent need for rigorous evaluation of e-cigarettes health effects to ensure evidence-based public health policies.

Malignant gliomas are the most common primary brain cancer diagnosed and still carry a poor prognosis despite aggressive multimodal management. Despite the continued advances in immunotherapy for other cancer types, however, there remain no FDA approved immunotherapies for cancers such as glioblastoma. OF the many approaches being explored, cancer vaccine programs are undergoing a renaissance due to the technological advances and personalized nature of their contemporary design. Neoantigen vaccines are a form of immunotherapy involving the use of DNA, mRNA, and proteins derived from non-synonymous mutations identified in patient tumor tissue samples to stimulate tumor-specific T-cell reactivity leading to enhance tumor targeting. In the last several years, the study of neoantigens as a therapeutic target has increased, with the routine workflow implementation of comprehensive next generation sequencing and in silico peptide binding prediction algorithms. Several neoantigen vaccine platforms are being evaluated in clinical trials for malignancies including melanoma, pancreatic cancer, breast cancer, lung cancer, and glioblastoma, among others. In this review, we will review the concept of neoantigen discovery using cancer immunogenomics approaches in glioblastoma and explore the disease-specific issues being addressed in the design of effective personalized cancer vaccine strategies.

Background: Last decade, there has been an increasing interest in the use of electronic cigarette (e-cig) among the youths. E-cigs are promoted as a safer alternative to tobacco cigarettes, but the long term health effects of e-cigs are unknown. E-cig aerosols contain harmful and potentially harmful substances, i.e., nicotine, ultrafine particles, flavoring agents, carbonyl compound, heavy metals, and carcinogens. In addition to containing reactive oxygen species (ROS), e-cig aerosols can also increase the cellular ROS production potentially leading to further alterations in the antioxidant system and its regulators. A recent in-vitro study from our laboratory showed that e-cigs aerosols can suppress the cellular antioxidant defenses and induced oxidative DNA damage in human epithelial cells. The effects of e-cig aerosols on antioxidant and detoxifying enzymes and its regulators are unknown. Aims: To investigate whether exposure to EC aerosol extracts alters the expression of antioxidant and detoxifying enzymes in human epithelial cells. Methods: Human oral epithelial cancer (UM-SCC-1) and non-cancer (POE9n) cell lines were exposed every other day for 2 weeks to e-cig aerosol. E-cig aerosols were prepared from two distinct e-cig brands (18 mg/ml of nicotine; tobacco flavor). Standard tobacco extracts (mainstream smoke, MS) were used as positive control. Gene and protein expression were quantified by RT-PCR and Western blotting respectively. Data were analyzed by Student's t-test and ANOVA. Results: Exposure to e-cig aerosol, but not to MS smoke, led to a decrease in NRF2 and SOD2 mRNA expression in both cell lines. A significant decrease in protein expression was also observed for NRF2 protein after exposure to e-cig aerosol. A significant increase in CYP1B1 was observed after exposure to e-cig aerosol and MS smoke. Conclusion: E-cig aerosol exposure decreases the expression of NRF2 a major regulator of the antioxidant response. This might put the cell under additional oxidative stress which can have major biological implications. E-cig aerosol exposure increases the expression of CYP1B1, a protein with a key role in the bioactivation of tobacco pro-carcinogens and the regulation of the redox homeostasis. Overall, our study suggests that e-cig aerosol not only carries high levels of ROS, but also alters the cellular detoxification and antioxidant system leading to a higher oxidative stress status that would be anticipated. Grant support: This work was supported by the National Institutes of Health, National Cancer Institute (R01CA242168). Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology, Oklahoma Tobacco Research Center (LQ). Citation Format: Vengatesh Ganapathy, Jimmy Manyanga, Lurdes Queimado. Differential expression of antioxidant and detoxifying enzymes induced by e-cigarette aerosol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2021.

Background Electronic cigarette (EC) aerosols contain unique compounds in addition to toxicants and carcinogens traditionally found in tobacco smoke. Studies are warranted to understand the public health risks of ECs. Objective The aim of this study was to determine the genotoxicity and the mechanisms induced by EC aerosol extracts on human oral and lung epithelial cells. Methods Cells were exposed to EC aerosol or mainstream smoke extracts and DNA damage was measured using the primer anchored DNA damage detection assay (q-PADDA) and 8-oxo-dG ELISA assay. Cell viability, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured using standard methods. mRNA and protein expression were evaluated by RT-PCR and western blot, respectively. Results EC aerosol extracts induced DNA damage in a dose-dependent manner, but independently of nicotine concentration. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts, as measured by q-PADDA. However, the levels of oxidative DNA damage, as indicated by the presence of 8-oxo-dG, a highly mutagenic DNA lesion, were similar or slightly higher after exposure to EC aerosol compared to mainstream smoke extracts. Mechanistically, while exposure to EC extracts significantly increased ROS, it decreased TAC as well as the expression of 8-oxoguanine DNA glycosylase (OGG1), an enzyme essential for the removal of oxidative DNA damage. Conclusions Exposure to EC aerosol extracts suppressed the cellular antioxidant defenses and led to significant DNA damage. These findings emphasize the urgent need to investigate the potential long-term cancer risk of exposure to EC aerosol for vapers and the general public.

Background and Aim: Are electronic cigarettes (ECs) safer than combustible tobacco? No one knows the answer yet. ECs are devices that deliver an aerosol from a heated mixture of propylene glycol, vegetable glycerin, and flavors, with or without nicotine. Short-term EC studies are mostly limited and inconclusive. Despite unknown long-term health effects, the use of ECs has increased alarmingly over the past decade. EC aerosols contain unique chemicals as well as other chemicals similar to those found in tobacco smoke. Tobacco smoking during cancer treatment has been associated with increased drug resistance and reduced overall survival rate. The awareness of the dangers of cigarette smoking and the paucity of studies evaluating the safety of ECs pose a dilemma for cancer patients who might be considering switching to ECs during chemotherapy. Here, we sought to examine the effects of EC aerosol extracts exposure on cell survival in oral cancer cells during cisplatin treatment. Methods: EC aerosol extracts were collected from two brands of ECs as previously described. Mainstream tobacco smoke (MS) extract was used as a positive control. To evaluate clonogenic survival after cisplatin treatment, oral cancer cells were exposed for 48 h to EC aerosol extracts at nicotine doses comparable to those observed in EC users. Next, cells were treated with both EC aerosol extracts and cisplatin (or vehicle-control) for another 48 h. After treatment, cells were counted and seeded in 6-well plates and colony formation was assessed two weeks later. Colonies were fixed with methanol followed by staining with 0.5% crystal violet in 25% methanol and counted manually. Only colonies with at least 50 cells were counted. Data were analyzed by Student's t-tests and one-way analysis of variance (ANOVA). Results: After cisplatin treatment, cells exposed to EC aerosol extracts showed a significant increase in clonogenic survival fraction compared to vehicle-control treated cells. Furthermore, cells exposed to EC aerosol extracts formed visibly larger colonies. An increase in clonogenic survival was also observed in MS extract-treated cells. Conclusions: Collectively, our data suggest that short-term exposure to EC aerosol can induce cisplatin resistance in oral cancer cells, thus allowing the cells to survive and form colonies. These findings are particularly important in paving the way to understanding whether ECs are a safer alternative for oral cancer patients undergoing chemotherapy. Citation Format: Jimmy Manyanga, Toral R. Mehta, Lurdes Queimado. Electronic cigarette aerosols increase clonogenic survival in oral cancer cells after cisplatin treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 80.

Background: E-cigarettes (ECs), also known as electronic nicotine delivery systems (ENDS) are battery-operated devices that deliver nicotine through inhaled aerosols. The health risks associated with EC use are unclear, but ECs have been promoted as a safer alternative to tobacco smoking and a smoking cessation aid. Currently, more than 13% of American high school students and 10% adults report using ECs. EC aerosols contain unique constituents (e.g. silicate beads, tin and flavorants), as well as other toxicants also present in tobacco smoke, including carcinogens and reactive oxygen species (ROS). The potential of EC aerosols to induce DNA damage has not been fully characterized. Aims: (1) To examine the effects of EC aerosol exposure in the levels of cellular reactive oxygen species (ROS); and (2) To evaluate the potential of EC aerosols to cause oxidative DNA damage. Methods: EC aerosols and mainstream smoke extracts were generated from distinct brands of ECs containing diverse nicotine concentrations and a reference combustible cigarette, in controlled conditions using a modified smoking apparatus. Human normal epithelial and oral cancer cell lines were exposed up to 2 weeks to diverse doses of EC aerosol extracts equivalent to 1 to 100 EC puffs. Levels of ROS were evaluated using the 2’,7’ -dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kit (Abcam). Overall DNA damage was quantified using two distinct assays q-PADDA and Comet assay. 8-Oxoguanine, one of the most common DNA lesions resulting from ROS, was quantified using a commercially available ELISA kit. Data were analyzed by Student’s t-test. Results: Exposure to EC aerosols caused a significant increase in ROS levels. Exposure to EC extracts induce significant DNA damage measurable by q-PADDA and Comet assay. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts. Yet, a significant increase in 8-oxoguanine lesions was observed after acute and long-term exposure to EC extracts compared to the control. Conclusion: Both short- and long-term exposure to EC aerosols can increase cellular ROS levels and cause significant DNA damage. Moreover, EC aerosols cause oxidative DNA lesions which are highly mutagenic, and can lead to tumor initiation and progression. Our study emphasizes the urgent need to further investigate the potential short- and long-term health effects of EC aerosols. Grant support: This work was supported by the Oklahoma Tobacco Research Center (LQ). Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Vengatesh Ganapathy, Jimmy Manyanga, Dehra McGuire, Daniel Brobst, Theodore Wagener, David Rubenstein, Ilangovan Ramachandran, Lurdes Queimado. Electronic cigarette aerosols increase cellular reactive oxygen species and induce significant oxidative DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 502. doi:10.1158/1538-7445.AM2017-502

Background: Cigarette smoking is the leading preventable cause of mortality in the world and the main risk factor for lung and head and neck cancer. E-cigarettes (ECs) are battery-operated devices that deliver nicotine via inhaled aerosols. Although ECs are marketed as a less harmful alternative to tobacco cigarettes and a smoking cessation aid, the health risks posed by exposure to EC aerosols are unknown. Nonetheless, the use of ECs has increased exponentially since 2003, with EC users reporting inhaling on average 200 puffs a day. EC aerosols have been reported to contain variable levels of genotoxins, including carcinogenic substances and reactive oxygen species (ROS). Some toxins in EC aerosols have been reported to reach the levels of similar to those in tobacco smoke. However, the genotoxicity of EC aerosols has not been characterized. Aims: (1) To determine the cytotoxicity and genotoxicity of short and long-term exposure to EC aerosols on human epithelial normal and cancer cell lines. (2) To evaluate whether exposure of EC aerosols modifies the cellular total antioxidant capacity (TAC). Methods: EC extracts were prepared from NJOY (12 or 18 mg/ml of nicotine) and Oakley eGo-T (0, 12 or 18 mg/ml of nicotine). Standard tobacco extracts were used for comparison. To assess the effects of short-term exposure, human epithelial normal (NuLi-1) and cancer (UD-SCC1) cell lines were exposed for 1 hour to various EC extract concentrations. To assess the effects of long-term exposure, cells lines were exposed every other day for 2 weeks to EC extracts. Cytotoxicity, DNA damage and TAC were evaluated at 1 h and 2 weeks. Cell viability was determined by MTT assay. DNA damage was quantified using the primer anchored DNA damage detection assay (PADDA). TAC was determined by the Antioxidant Assay Kit (Cayman). Data were analyzed by Student's t-test. Results: At the range of EC extract concentrations used in this study and expected to occur in EC users (1 to 100 puffs/5 L of blood), no cytotoxicity was observed for either normal or cancer cells. However, significant levels of DNA damage were observed in cancer cells exposed to 10 or more puffs/5 L of EC extracts and in normal cells exposed to100 puffs/5 L. Long-term exposure to EC extracts resulted in a significant decrease in TAC, a measure of free radical scavengers. Conclusion: Even short-term exposure to low levels of EC aerosols can cause significant DNA damage. Our study emphasizes the need to further investigate the carcinogenic potential of EC aerosols and highlights the importance of regulating EC use. Grant support: This work was supported by the Oklahoma Tobacco Research Center (LQ). Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Vengatesh Ganapathy, Jimmy Manyanga, Lacy Brame, David Rubenstein, Theodore L. Wagener, Ilangovan Ramachandran, Lurdes Queimado. Electronic cigarettes induce significant DNA damage and reduce cellular antioxidant levels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3593.