Cellular diversity through space and time: adding new dimensions to GBM therapeutic development.

The current median survival for glioblastoma (GBM) patients is only about 16 months, with many patients succumbing to the disease in just a matter of months, making it the most common and aggressive primary brain cancer in adults. This poor outcome is, in part, due to the lack of new treatment options with only one FDA-approved treatment in the last decade. Advances in sequencing techniques and transcriptomic analyses have revealed a vast degree of heterogeneity in GBM, from inter-patient diversity to intra-tumoral cellular variability. These cutting-edge approaches are providing new molecular insights highlighting a critical role for the tumor microenvironment (TME) as a driver of cellular plasticity and phenotypic heterogeneity. With this expanded molecular toolbox, the influence of TME factors, including endogenous (e.g., oxygen and nutrient availability and interactions with non-malignant cells) and iatrogenically induced (e.g., post-therapeutic intervention) stimuli, on tumor cell states can be explored to a greater depth. There exists a critical need for interrogating the temporal and spatial aspects of patient tumors at a high, cell-level resolution to identify therapeutically targetable states, interactions and mechanisms. In this review, we discuss advancements in our understanding of spatiotemporal diversity in GBM with an emphasis on the influence of hypoxia and immune cell interactions on tumor cell heterogeneity. Additionally, we describe specific high-resolution spatially resolved methodologies and their potential to expand the impact of pre-clinical GBM studies. Finally, we highlight clinical attempts at targeting hypoxia- and immune-related mechanisms of malignancy and the potential therapeutic opportunities afforded by single-cell and spatial exploration of GBM patient specimens.

CSF1R-dependent macrophages in the salivary gland are essential for epithelial regeneration after radiation-induced injury.

The salivary glands often become damaged in individuals receiving radiotherapy for head and neck cancer, resulting in chronic dry mouth. This leads to detrimental effects on their health and quality of life, for which there is no regenerative therapy. Macrophages are the predominant immune cell in the salivary glands and are attractive therapeutic targets due to their unrivaled capacity to drive tissue repair. Yet, the nature and role of macrophages in salivary gland homeostasis and how they may contribute to tissue repair after injury are not well understood. Here, we show that at least two phenotypically and transcriptionally distinct CX3CR1+ macrophage populations are present in the adult salivary gland, which occupy anatomically distinct niches. CD11c+CD206-CD163- macrophages typically associate with gland epithelium, whereas CD11c-CD206+CD163+ macrophages associate with blood vessels and nerves. Using a suite of complementary fate mapping systems, we show that there are highly dynamic changes in the ontogeny and composition of salivary gland macrophages with age. Using an in vivo model of radiation-induced salivary gland injury combined with genetic or antibody-mediated depletion of macrophages, we demonstrate an essential role for macrophages in clearance of cells with DNA damage. Furthermore, we show that epithelial-associated macrophages are indispensable for effective tissue repair and gland function after radiation-induced injury, with their depletion resulting in reduced saliva production. Our data, therefore, provide a strong case for exploring the therapeutic potential of manipulating macrophages to promote tissue repair and thus minimize salivary gland dysfunction after radiotherapy.

Use of a Novel Hypercrosslinked Carbohydrate Scaffold for Vocal Fold Medialization in an Ovine Model.

Objectives: Vocal fold medialization is commonly performed for glottic insufficiency and vocal fold immobility. Currently available materials are temporary injectables or synthetic implants. Acellular scaffolds may allow vocal fold augmentation with autologous tissue via host cell migration. The purpose of this investigation was to evaluate the use of a novel carbohydrate scaffold as a medialization implant. Study Design: Animal model. Setting: Academic medical center. Methods: Unilateral type I medialization thyroplasty was performed in 3 Dorper cross ewes using a hypercrosslinked carbohydrate polymer (HCCP) scaffold. Animals were monitored for 4 weeks for general well-being, dyspnea, and weight loss. The animals were euthanized at 4 weeks and the larynges harvested. Histologic evaluation was performed to assess for adverse tissue reaction, migration, degradation, and biocompatibility. Results: No adverse events were reported. No animals lost weight or displayed evidence of dyspnea. Histology demonstrated ingrowth of host cells and neovascularization with minimal peri-implant inflammatory reaction. Cellular ingrowth into the scaffold was predominately made up of fibroblasts and early inflammatory cells. Scaffold shape was grossly maintained as it underwent degradation and replacement with host tissue. Migration of the implant material was not observed. Conclusion: Vocal fold medialization in an ovine model with an HCCP scaffold resulted in the ingrowth of host cells with minimal peri-implant inflammation. Scaffold shape was maintained without evidence of migration as it underwent replacement with host tissue. Further research is required to assess long-term efficacy in comparison to currently available implants.

Subcutaneous choriocarcinomas in captive Amargosa voles (Microtus californicus scirpensis).

Spontaneous choriocarcinomas are rare, highly vascular, malignant trophoblastic tumors that occur in humans and animals. This report describes the unusual spontaneous presentation of 4 choriocarcinomas within the subcutaneous tissues of 4, multiparous but nongravid, Amargosa voles (Microtus californicus scirpensis) from a captive breeding colony. Two subcutaneous neoplasms were composed of multifocal discohesive and infiltrative aggregates of medium to large trophoblasts and cytotrophoblasts within a fibrovascular stroma. Neoplastic cells were associated with variably sized thrombi and cavitary areas of hemorrhage and necrosis. Two subcutaneous tumors were predominantly composed of expansile, blood-filled, cystic spaces lined by neoplastic cytotrophoblasts and occasionally contained medium to large trophoblasts. Trophoblasts and cytotrophoblasts were positive for pancytokeratin and cytokeratin 8/18, negative for alpha-fetoprotein, and contained intracytoplasmic Periodic acid-Schiff (PAS)-positive glycogen in all 4 tumors. In species with hemochorial placentation, migration of trophoblasts into maternal circulation with embolization to distant nonreproductive tissues occurs and may explain the unusual subcutaneous distribution of these 4 tumors. The 2 multiloculated paucicellular tumors may represent an early stage of neoplastic transformation. To the authors' knowledge, this is the first report characterizing choriocarcinomas in extrareproductive sites in rodents.

The Role of Supporting Cell Populations in Satellite Cell Mediated Muscle Repair.

Skeletal muscle has a high capacity to repair and remodel in response to damage, largely through the action of resident muscle stem cells, termed satellite cells. Satellite cells are required for the proper repair of skeletal muscle through a process known as myogenesis. Recent investigations have observed relationships between satellite cells and other cell types and structures within the muscle microenvironment. These findings suggest that the crosstalk between inflammatory cells, fibrogenic cells, bone-marrow-derived cells, satellite cells, and the vasculature is essential for the restoration of muscle homeostasis. This review will discuss the influence of the cells and structures within the muscle microenvironment on satellite cell function and muscle repair.

Birth Outcomes Associated With E-Cigarette and Non-E-Cigarette Tobacco Product Use During Pregnancy: An Examination of PATH Data Waves 1-5.

OBJECTIVES: To examine associations of prenatal e-cigarette use to pregnancy and birth outcomes. METHODS: Currently pregnant women (n = 1 037) from Waves 1 through 4 of the Population Assessment of Tobacco and Health Study who had pregnancy or live birth outcome data in a subsequent wave (Waves 2-5; 2013 to 2019). Weighted bivariate and multivariable models\ examined associations between past 30-day tobacco use assessed during pregnancy (any past 30-day e-cigarette use, any past 30-day non-e-cigarette tobacco use, or no past 30-day tobacco use) with adverse pregnancy (miscarriage, abortion, ectopic or tubal pregnancy, stillbirth) and birth outcomes (preterm birth, low birth weight, birth defect, placenta previa, placental abruption, pre-eclampsia) reported in the subsequent wave. RESULTS: Approximately 1% of pregnant women reported past 30-day exclusive e-cigarette use and 3.2% used e-cigarettes and one other tobacco product. Compared to no tobacco use, past 30-day e-cigarette use (exclusive or use with another tobacco product) during pregnancy was not associated with increased odds of an adverse pregnancy or birth outcome in bivariate or multivariable models. Past 30-day non-e-cigarette tobacco use was associated with increased odds of an adverse pregnancy outcome in multivariable models, but not an adverse live birth outcome. Compared to past 30-day cigarette use, past 30-day e-cigarette use during pregnancy was not associated with lowered odds of a birth or pregnancy outcome. CONCLUSIONS: E-cigarette use during pregnancy is rare. Understanding the positive and negative impacts of pre-natal e-cigarette use on women's health may guide public health messaging campaigns. IMPLICATIONS: Results showed that past 30-day e-cigarette use during pregnancy was low, with cigarette smoking remaining the most prevalent form of tobacco use during pregnancy. Current e-cigarette use during pregnancy used either exclusively or with another tobacco product, was not associated with increased risk of an adverse pregnancy, or birth outcome. A small sample size of e-cigarette users and limited information on quantity and frequency of e-cigarette use before and during pregnancy may limit conclusions. Healthcare providers may use this information when discussing the harms and consequences associated with e-cigarette and tobacco use during pregnancy.

Exploring glioblastoma stem cell heterogeneity: Immune microenvironment modulation and therapeutic opportunities.

Despite its growing use in cancer treatment, immunotherapy has been virtually ineffective in clinical trials for gliomas. The inherently cold tumor immune microenvironment (TIME) in gliomas, characterized by a high ratio of pro-tumor to anti-tumor immune cell infiltrates, acts as a seemingly insurmountable barrier to immunotherapy. Glioma stem cells (GSCs) within these tumors are key contributors to this cold TIME, often functioning indirectly through activation and recruitment of pro-tumor immune cell types. Furthermore, drivers of GSC plasticity and heterogeneity (e.g., reprogramming transcription factors, epigenetic modifications) are associated with induction of immunosuppressive cell states. Recent studies have identified GSC-intrinsic mechanisms, including functional mimicry of immune suppressive cell types, as key determinants of anti-tumor immune escape. In this review, we cover recent advancements in our understanding of GSC-intrinsic mechanisms that modulate GSC-TIME interactions and discuss cutting-edge techniques and bioinformatics platforms available to study immune modulation at high cellular resolution with exploration of both malignant (i.e., GSC) and non-malignant (i.e., immune) cell fractions. Finally, we provide insight into the therapeutic opportunities for targeting immunomodulatory GSC-intrinsic mechanisms to potentiate immunotherapy response in gliomas.

Synthesis and Evaluation of Functionalized Aryl and Biaryl Isothiocyanates against Human MCF-7 Cells.

Organic isothiocyanates (ITCs) are a class of anticancer agents which naturally result from the enzymatic degradation of glucosinolates produced by Brassica vegetables. Previous studies have demonstrated that the structure of an ITC impacts its potency and mode(s) of anticancer properties, opening the way to preparation and evaluation of synthetic, non-natural ITC analogues. This study describes the preparation of a library of 79 non-natural ITC analogues intended to probe further structure-activity relationships for aryl ITCs and second-generation, functionalized biaryl ITC variants. ITC candidates were subjected to bifurcated evaluation of antiproliferative and antioxidant response element (ARE)-induction capacity against human MCF-7 cells. The results of this study led to the identification of (1) several key structure-activity relationships and (2) lead ITCs demonstrating potent antiproliferative properties.

Common and Not-So-Common Pathologic Findings of the Gastrointestinal Tract of Rhesus and Cynomolgus Macaques.

Rhesus and cynomolgus macaques are the most frequently used nonhuman primate (NHP) species for biomedical research and toxicology studies of novel therapeutics. In recent years, there has been a shortage of laboratory macaques due to a variety of competing factors. This was most recently exacerbated by the surge in NHP research required to address the severe acute respiratory syndrome (SARS)-coronavirus 2 pandemic. Continued support of these important studies has required the use of more varied cohorts of macaques, including animals with different origins, increased exposure to naturally occurring pathogens, and a wider age range. Diarrhea and diseases of the gastrointestinal tract are the most frequently occurring spontaneous findings in macaques of all origins and ages. The purpose of this review is to alert pathologists and scientists involved in NHP research to these findings and their impact on animal health and study endpoints, which may otherwise confound the interpretation of data generated using macaques.

A novel non-human primate model of Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a severe hypomyelinating disorder of the central nervous system (CNS) linked to mutations in the proteolipid protein-1 (PLP1) gene. Although there are multiple animal models of PMD, few of them fully mimic the human disease. Here, we report three spontaneous cases of male neonatal rhesus macaques with the clinical symptoms of hypomyelinating disease, including intention tremors, progressively worsening motor dysfunction, and nystagmus. These animals demonstrated a paucity of CNS myelination accompanied by reactive astrogliosis, and a lack of PLP1 expression throughout white matter. Genetic analysis revealed that these animals were related to one another and that their parents carried a rare, hemizygous missense variant in exon 5 of the PLP1 gene. These animals therefore represent the first reported non-human primate model of PMD, providing a novel and valuable opportunity for preclinical studies that aim to promote myelination in pediatric hypomyelinating diseases.

Predictors of Subcutaneous Injection Site Reactions to Sustained-Release Buprenorphine in Rhesus Macaques (Macaca mulatta).

Subcutaneous injection site reactions to sustained-release buprenorphine hydrochloride (Buprenorphine SR) in macaques have been reported in only a single case report. In the current study, we evaluated the incidence rate and predictors of buprenorphine SR reactions in the subcutaneous tissue of rhesus macaques (Macaca mulatta) based on retrospective review of macaque buprenorphine SR injection records. Potentially predictive variables were identified with logistic regression modeling and were evaluated using model selection based on Akaike information criterion. Record review revealed sub- cutaneous tissue reactions occurred in 52 (3%) of 1559 injections and were noted between 4 and 311 d after injection. Model selection showed that body weight and MHC allele Mamu-B*29 were the best predictors of subcutaneous reactions. Based on these results, we recommend consideration of potential risk factors prior to the administration of buprenorphine SR to a rhesus macaque. In addition, the authors advise that using the highest concentration of buprenorphine SR available may reduce injection site reaction rates due to the injection of less copolymer.

Differences in reasons for little cigar/cigarillo use across white and black/African American young adult users.

INTRODUCTION: The prevalence of little cigar and cigarillo (LCC) use among young adults is high. Research shows there are racial/ethnic differences in this prevalence, with Black/African American users more likely to report current LCC use. Given these discrepancies in LCC use, the present study aimed to assess potential differences in reasons for LCC use between White and Black/African American young adult ever and past 30-day users. METHODS: Participants were White (n = 2150), and Black/African American (n = 308) young adults (aged 18 to 24) recruited from Amazon Mechanical Turk who completed an online survey of tobacco use (December 2018-January 2019). LCC users were asked eight reasons for using LCCs. RESULTS: Flavoring was cited as the most popular reason for LCC use among White ever users while affordability was the most popular among Black/African American ever users. Adjusted logistic regressions among ever users revealed that Black/African American respondents (vs White) were more likely to use LCCs because of their affordability, the perception that LCCs are less harmful than cigarettes, and because of LCC advertising appeal. Among past 30-day users, adjusted logistic regression models showed that Black/African American respondents (vs White) were more likely to use LCCs because of their affordability. CONCLUSIONS: Findings align with previous studies showing that LLCs are more heavily marketed in predominantly Black/African American communities. Prevention efforts should account for racial differences in reasons for use in message development.

Colon volvulus in rhesus macaques (Macaca mulatta) at the Oregon National Primate Research Center-Retrospective analysis.

Colonic volvulus is an uncommon, often life-threatening condition, in non-human primates. Twenty-six cases of colonic volvulus in rhesus macaques (Macaca mulatta) were identified in necropsy records spanning 38 years at the Oregon National Primate Research Center (ONPRC). This report represents the largest collection of colonic volvuli in rhesus macaques.

Urothelial carcinoma in the urinary bladder of a Japanese macaque (Macaca fuscata).

Tumors of urinary origin are infrequently reported in non-human primates. Urothelial carcinoma involving the urinary bladder was diagnosed in an adult female Japanese macaque that extended transmurally to the uterus and cervix. To our knowledge, this is the first report of a primary cystic urothelial carcinoma in a Japanese macaque.

Youth Vaping and Tobacco Use in Context in the United States: Results From the 2018 National Youth Tobacco Survey.

INTRODUCTION: According to the National Youth Tobacco Survey (NYTS), youth e-cigarette use (vaping) rose between 2017 and 2018. Frequency of vaping and concurrent past 30-day (p30d) use of e-cigarettes and tobacco products have not been reported. METHODS: We analyzed the 2018 NYTS (N = 20 189) for vaping among all students (middle and high school; 6-12th grades; 9-19 years old) by frequency of vaping, exclusive vaping, p30d poly-product use (vaping and use of one or more tobacco product), and any past tobacco product use. RESULTS: In 2018, 81.4% of students had not used any tobacco or vapor product in the p30d, and 86.2% had not vaped in the p30d. Among all students, of the 13.8% vaped in the p30d, just over half vaped on ≤5 days (7.0%), and roughly a quarter each vaped on 6-19 days (3.2%) and on 20+ days (3.6%). Almost three quarters of p30d vapers (9.9%) reported past or concurrent tobacco use and the remainder (3.9%) were tobacco naïve. 2.8% of students were tobacco naïve and vaped on ≤5 days; 0.7% were tobacco-naïve and vaped on 6-19 days, and 0.4% were tobacco-naïve and vaped on 20+ days. CONCLUSIONS: Vaping increased among US youth in 2018 over 2017. The increases are characterized by patterns of low p30d vaping frequency and high poly-product use, and a low prevalence of vaping among more frequent but tobacco naïve vapers. IMPLICATIONS: Results underscore the importance of including the full context of use patterns. The majority of vapers (60.0%-88.9% by use frequency) were concurrent p30d or ever tobacco users. About 4% of students were tobacco naïve and vaped in the p30d, but few (0.4%) vaped regularly on 20 or more days. Reporting youth vaping data with frequency and tobacco product co-use will give public health decision-makers the best possible information to protect public health.

Adolescent Susceptibility to E-Cigarettes: An Update From the 2018 National Youth Tobacco Survey.

PURPOSE: Examine correlates of e-cigarette susceptibility among adolescents. DESIGN: Secondary data analyses using the 2018 National Youth Tobacco Survey, excluding participants under 12 and over 17. SETTING: United States middle and high schools. SUBJECTS: Never e-cigarette users (n = 12,439) ages 12-17. MEASURES: Relationships between e-cigarette susceptibility and age, sex, race/ethnicity, ever tobacco use, perceived ease of purchasing tobacco products, perceived harm, relative addictiveness, household use of e-cigarettes/tobacco were examined. ANALYSIS: Odds of susceptibility were modeled with weighted multivariable logistic regressions. RESULTS: Thirty-five percent (unweighted n = 4,436) of adolescents were susceptible to e-cigarettes. Adolescents who were female (aOR = 1.2), Hispanic (aOR = 1.3), perceived e-cigarettes as anything less than "a lot of harm" (aOR = 2.2-4.9) and "easy" to purchase (aOR = 1.4), had ever used combustible tobacco (aOR = 2.9), or reported household use of e-cigarettes (aOR = 1.5) were susceptible. Non-Hispanic black respondents (vs. non-Hispanic white; aOR = 0.72) had significantly lower odds of susceptibility to e-cigarettes. CONCLUSION: In the 2018 NYTS adolescent sample, perceptions of harm and ease of tobacco product purchase appear to be significantly related to higher odds of e-cigarette susceptibility, in addition to other demographic factors. Longitudinal data, particularly cohort data following adolescents from susceptible to actual or no use, are needed to assess predictors of e-cigarette use initiation.

Two cases of cryptococcus gattii infection in the rhesus macaque (Macaca Mulatta).

Cryptococcus gattii was diagnosed in two female indoor-housed rhesus macaques. Gross and histopathologic findings included an isolated pulmonary cryptococcoma in a non-SIV-infected macaque and disseminated disease centered on the lungs of an SIV-infected macaque. Fungal yeast were positive with special stains, and the diagnoses were confirmed with a lateral flow assay and PCR.

Menthol and Mint Cigarettes and Cigars: Initiation and Progression in Youth, Young Adults and Adults in Waves 1-4 of the PATH Study, 2013-2017.

INTRODUCTION: This study examined in youth (12-17 years), young adults (18-24 years), and adults (25+ years): (1) the prevalence of the first menthol cigarette and menthol/mint cigar use among new tobacco users; (2) association between the first menthol/mint use, subsequent tobacco use, and nicotine dependence ~1 year later compared with the first non-menthol/mint use. AIMS AND METHODS: Longitudinal analysis of data from Waves 1 to 4 of the Population Assessment of Tobacco and Health (PATH) Study (2013-2017; 10 086 youth and 21 281 adults). Main outcome measures were past 12-month and past 30-day cigarette and cigar use, and nicotine dependence. RESULTS: Youth and young adult new cigarette users are more likely to smoke a menthol cigarette or indicate that they do not know the flavor compared with adults aged 25+. A greater proportion of adults aged 25+ first used menthol/mint-flavored cigars (13.4%) compared with youth (8.5%) and young adults (7.4%). Among young adults, first use of a menthol cigarette is associated with past 12-month use of cigarettes at the subsequent wave and first use of any menthol/mint-flavored cigars is associated with past 30-day use of these products at the subsequent wave in both youth and young adults. In youth and adults, there were no significant relationships between first use of a menthol/mint cigarette or cigar and nicotine dependence scores at a subsequent wave in multivariable analyses. CONCLUSIONS: The first use of menthol/mint cigarettes and cigars is associated with subsequent cigarette and cigar use in young people aged 12-24. IMPLICATIONS: This study examined the relationship between initiation with menthol cigarettes and menthol/mint cigars, subsequent tobacco use, and nicotine dependence in US youth, young adults, and adults who participated in Waves 1-4 of the Population Assessment of Tobacco and Health study. New use of menthol cigarettes was associated with greater past 12-month cigarette use in young adults and new use of menthol/mint-flavored cigars was associated with greater past 30-day cigar use in youth and young adults compared with non-menthol use. Initiation with menthol/mint cigarette and cigar products may lead to subsequent use of those products.

Association of Electronic Nicotine Delivery System Use With Cigarette Smoking Progression or Reduction Among Young Adults.

Importance: The prevalence of electronic nicotine delivery systems (ENDS) use, including e-cigarettes, among US young adults (YAs) has raised questions about how these products may affect future tobacco and nicotine use among YAs. Given this prevalence and that young adulthood is a critical period for the establishment of tobacco and nicotine use, it is important to consider the association between ENDS use and cigarette smoking specifically in this age group. Objective: To examine whether ENDS use frequency or intensity is associated with changes in cigarette smoking among US YA ever smokers during 1 year. Design, Setting, and Participants: This cohort study used 3 waves of data (2013-2014, 2014-2015, and 2015-2016) from the Population Assessment of Tobacco and Health (PATH) Study, an ongoing longitudinal cohort study of adults and youth. Unweighted 1:6 propensity score matching was used to match participants on wave 1 risk factors for ENDS use at wave 2. The changes in smoking between wave 2 and wave 3 were assessed using the matched sample. In total, 1096 ENDS-naive, ever cigarette-smoking YAs (18-24 years of age) at wave 1 who participated in wave 2 and wave 3 and who had complete data in the PATH Study were included in the analyses, which were conducted from August 2018 to October 2019. Exposures: Never ENDS use (n = 987), any previous 30-day ENDS use (n = 109), 1 to 5 days of ENDS use in the previous 30 days (n = 75), and 6 or more days ENDS use in the previous 30 days at wave 2 (n = 34). Main Outcomes and Measures: The analytic sample was selected using multiple variables based on peer-reviewed literature supporting associations with ENDS use. The main outcomes-changes in cigarette smoking behavior between wave 2 and wave 3-were defined using 2 measures: (1) change in smoking frequency, defined as the number of smoking days in the previous 30 days at wave 3 vs wave 2, and (2) change in smoking intensity, defined as the number smoking days in the previous 30 days multiplied by the mean number of cigarettes consumed on smoking days at wave 3 vs wave 2. Results: The present cohort analyses included 1096 YA ever smokers who were ENDS naive at wave 1. The majority of the sample were women (609 [55.6%]) and White individuals (698 [63.7%]), and the mean (SD) age was 21.4 (1.9) years. In wave 1, 161 YAs (14.7%) were daily smokers in the previous 30 days. After propensity score matching, no statistically significant associations were observed between any definition of wave 2 ENDS use and changes in either the frequency or intensity of smoking at wave 3. Conclusions and Relevance: In this cohort study of US YA ever smokers, ENDS use was not associated with either decreased or increased cigarette smoking during a 1-year period. However, it is possible that the rapidly evolving marketplace of vaping products may lead to different trajectories of YA cigarette and ENDS use in the future.

Impact of flavors and humectants on waterpipe tobacco smoking topography, subjective effects, toxicant exposure and intentions for continued use.

INTRODUCTION: The present study examined how the lack of characterising flavours and low levels of humectants may affect users' waterpipe tobacco (WT) smoking topography, subjective effects, toxicant exposure and intentions for continued use. METHODS: 89 WT smokers completed four ad libitum smoking sessions (characterising flavor/high humectant (+F+H); characterising flavor/low humectant (+F-H); no characterising flavor/high humectant (-F+H); no characterising flavor/low humectant (-F-H)) in a randomised cross-over design. WT was commercially available; same brand but nicotine levels were not held constant. A subsample (n=50) completed a standardised, 10-puff session preceding ad libitum smoking. Participants completed questionnaires, exhaled carbon monoxide (eCO) testing and provided blood samples for plasma nicotine. Smoking topography was measured throughout the session. Post hoc analyses showed that -F+H and -F-H did not differ significantly in humectant levels. Therefore, these groups were collapsed in analyses (-F-H). RESULTS: WT smokers reported significantly greater satisfaction, liking, enjoyment and greater intentions for continued use when smoking +F+H compared with other WT products, with -F-H receiving the lowest ratings. Significant differences in topography were observed during standardised and ad libitum sessions, with the -F-H preparation leading to greater total inhaled volume and eCO boost, but lower nicotine boost compared with +F+H (all p<0.05). DISCUSSION: The findings demonstrate the importance of flavours and humectants on improving WT smoking experience and increasing the likelihood that users will want to initiate and continue smoking. Moreover, it demonstrates that flavours and humectants influence smoking behaviour and toxicant exposure in some unexpected ways that are important for regulatory efforts.