pmc0417615848Arch ToxicolArch ToxicolArchives of toxicology0340-57611432-0738397865901286047110.1007/s00204-024-03932-xNIHMS2141293ArticleE-cigarettes increase the risk of adenoma formation in murine colorectal cancer modelhttp://orcid.org/0000-0001-8207-6401SayedIbrahim M.12ChakrabortyAnirban3InouyeKaili1DuganLeanne1TocciStefania2AdvaniIra4ParkKenneth4GaboyanSamvel4KasaraneniNikita4MaBenjamin4HazraTapas K.3DasSoumita12Crotty AlexanderLaura E.45Department of Pathology, University of California, San Diego, CA 92093, USADepartment of Biomedical and Nutritional Sciences, Zuckerberg College of Health Sciences, University of Massachusetts Lowell, Lowell, MA 01854, USADepartment of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555, USADepartment of Medicine, University of California, San Diego, CA 92093, USAMedicine Service, VA San Diego Healthcare System, San Diego, CA 92161, USA

Author contributions IMS, SD, and LCA designed the study. IMS, KI, LD, ID, SG, NK, BA, and KP performed animal experiments; AC performed western blot and LA-PCR assay; ST helped in IF imaging, IMS, AC, HTK, SD, and LCA analyzed data; HKT for resources of DNA damage assays, SD and LCA supervised the research. IMS wrote the original draft;, SD and LCA edited and revised the manuscript. All authors edited and agreed on the final version of the manuscript.

Ibrahim M. Sayed, Ibrahim_ibrahim@uml.edu, Soumita Das, soumita_das@uml.edu, Laura E. Crotty Alexander, lca@ucsd.edu2612026320250912025311202699312231236

E-cigarettes (E.cigs) cause inflammation and damage to human organs, including the lungs and heart. In the gut, E.cig vaping promotes inflammation and gut leakiness. Further, E.cig vaping increases tumorigenesis in oral and lung epithelial cells by inducing mutations and suppressing host DNA repair enzymes. It is well known that cigarette (cig) smoking increases the risk of colorectal cancer (CRC). To date, it is unknown whether E.cig vaping impacts CRC development. A mouse model of human familial adenomatous polyposis (CPC-APC) was utilized wherein a mutation in the adenomatous polyposis coli (APC) gene, CDX2-Cre-APCMin/+, leads to the development of colon adenomas within 16 weeks. Mice were exposed to air (controls), E.cig vaping, cig, or both (dual exposure). After 4 weeks of 2 h exposures per day (1 h of each for dual exposures), the colon was collected and assessed for polyp number and pathology scores by microscopy. Expression of inflammatory cytokines and cancer stem cell markers were quantified. DNA damage such as double-strand DNA breaks was evaluated by immunofluorescence, western blot, and gene-specific long amplicon qPCR. DNA repair enzyme levels (NEIL-2, NEIL-1, NTH1, and OGG1) were quantified by western blot. Proliferation markers were assessed by RT-qPCR and ELISA. CPC-APC mice exposed to E.cig, cig, and dual exposure developed a higher number of polyps compared to controls. Inflammatory proteins, DNA damage, and cancer stemness markers were higher in E-cig, cig, and dual-exposed mice as well. DNA damage was found to be associated with the suppression of DNA glycosylases, particularly with NEIL-2 and NTH1. E.cig and dual exposure both stimulated cancer cell stem markers (CD44, Lgr-5, DCLK1, and Ki67). The effect of E.cigs on polyp formation and CRC development was less than that of cigs, while dual exposure was more tumorigenic than either of the inhalants alone. E.cig vaping promotes CRC by stimulating inflammatory pathways, mediating DNA damage, and upregulating transcription of cancer stem cell markers. Critically, combining E.cig vaping with cig smoking leads to higher levels of tumorigenesis. Thus, while the chemical composition of these two inhalants, E.cigs and cigs, is highly disparate, they both drive the development of cancer and when combined, a highly common pattern of use, they can have additive or synergistic effects.

E.cigColorectal cancerCigarette smokingDual useCPC-APC miceDNA damageDNA glycosylaseInflammationStemness markersIntroduction

E-cigarettes (E.cigs) are a non-combustible form of tobacco and are especially popular among youth. E-cigs were first introduced in the mid-2000s, and have become widespread in the US, especially among adolescents. Approximately 10 million US adults and 2 million kids are active E.cig users (McCarthy 2016; Mirbolouk et al. 2018; Regan et al. 2013; Gentzke et al. 2019). E.cig liquids (e-liquids) consist of vehicle solvents, such as propylene glycol (PG) and glycerin, commonly called vegetable glycerin (VG), combined with nicotine, which are heated and aerosolized within e-devices. A large variety of flavoring chemicals (~ 7000) are added to e-liquids to increase appeal (Zhu et al. 2014). However, e-cigarette use has been linked to several adverse health effects, including lung injuries such as EVALI (King et al. 2020; Shinbashi and Rubin 2020). Chronic inhalation of E.cig affects the inflammatory states in the lungs and circulation and can increase the risk of infectious diseases (Sayed et al. 2021a). Our recent study showed that E.cig vaping disrupts gut barrier integrity and induces inflammation and gut leakiness (Sharma et al. 2021).

CRC is the second leading cause of death in USA (Siegel et al. 2023). Several factors can contribute to the development of CRC, such as genetic mutations, environmental factors, lifestyles, and microbial dysbiosis (Sayed et al. 2021b). CRCs are primarily caused by the loss of tumor-suppressor genes, such as APC (Morin et al. 1997), and almost 80% of CRC cases are due to APC mutations (Zhang and Shay 2017). The loss of APC gene is the cause of familial adenomatous polyposis (FAP) (Groden et al. 1991). CPC-APC (CDX2-Cre-APCmin with a mutation in the APC gene) mice are a mouse model of FAP and CRC that spontaneously develop multiple colonic polyps (adenomas) and rectal bleeding at around 16 weeks (Hinoi et al. 2007). Cigarette smoke exposure increases the development of adenomas in APCMin/+ mice by altering intestinal permeability (Wang et al. 2021), and induced DNA damage in the colon (Diergaarde et al. 2003; Sarebø et al. 2006; Lüchtenborg et al. 2005). While traditional cigarette smoking has been linked to the development of CRC (Liang et al. 2009; Botteri et al. 2020; Tsoi et al. 2009), there is no direct evidence linking E.cig vaping to CRC development.

Toxicological studies have reported the presence of heavy metals, formaldehyde, and acrolein in E.cig aerosols, commonly called vapor (Salamanca et al. 2018). In addition, PG, VG, flavorings, and contaminants can be toxic (Perez and Crotty Alexander 2020; Bozier et al. 2020). E.cig vapor has been shown to induce DNA damage and accumulative mutations (Ganapathy et al. 2017; Yu et al. 2016), which were comparable to or slightly higher than the effect mediated by mainstream smoke extracts (Ganapathy et al. 2017). Using 3D human and murine organoids and 2D enteroid-derived monolayers, we have shown that exposure to E.cig vapor increased the bacterial translocation inside the gut, upregulated inflammatory responses, and altered DNA damage responses (Sharma et al. 2021).

Following DNA damage, such as oxidation, deamination, and alkylation, the host base excision repair (BER) proteins/DNA glycosylases can excise the damaged based and the downstream steps correct the DNA damage to prevent accumulation of the DNA mutation and development of CRC (Krokan and Bjørås 2013; Meira et al. 2008). Suppression and downregulation of DNA glycosylases, especially NEIL-2 and NEIL-1, have been associated with the development of lung cancer (Dey et al. 2012). Our studies showed that the gastric cancer-associated microbes Helicobacter pylori and CRC-associated microbe Fusobacterium nucleatum downregulate epithelial DNA glycosylase NEIL-2, increase inflammation, and lead to the accumulation of DNA damage and cancers (Sayed et al. 2020a, 2020b). Exposure to E.cig vapor causes a suppression of OGG1 in oral and lung epithelial cells resulting in the accumulation of reactive oxygen species, 8-hydroxy-2’-deoxyguanosine (8-oxo-dG) bases which are genotoxic (Ganapathy et al. 2017). However, the effect of E.cig exposure on other DNA glycosylases and in the colon has not been studied.

It is thought that switching from conventional tobacco to E.cigs can bypass the harmful effects of conventional smoking (O’Loughlin et al. 2016); however, dual use of both cigarettes and E.cigs remain common. Previous studies showed that a combined effect of E.cig and cigarette smoke was worse than the effect of a single exposure. The combined effect caused more toxicity, and enhanced inflammatory responses, compared to the single exposure (Hamon et al. 2024). The effect of single exposure on the colon was reported; both E.cig and cigarette smoke disrupted epithelial tight junctions and induced gut leakiness (Sharma et al. 2021; Wang et al. 2021). However, the combined effect on the colon, more specifically on the development of CRC, has not been reported.

In this study, we assessed the impact of E.cig vapor inhalation alone and in combination with cigarette smoke on the development and progression of CRC. We exposed CPC-APC mice to E.cig vapor, cigarette smoke, and both inhalants (dual exposure), or air (control), and assessed CRC development via tumor number, tumor size, inflammation, DNA damage, and intestinal stemness markers. We also assessed the damaging effects of E.cigs on genomic DNA and on host DNA repair enzymes.

MethodsAnimals and inhalant exposures

CPC-APC (CDX2-Cre-APCmi) mice were housed, bred, enrolled in the experimental design, and euthanized according to the University of California San Diego Institutional Animal Care and Use Committee (IACUC) policies under animal protocol numbers (s18086 and s16021). Prior to randomization at the beginning of all studies, all mice of the same sex were group housed in a large sterile container for 2 h, to allow free mixing of microbiota amongst individuals.

CPC-APC mice of both genders, aged 9–12 weeks, were exposed for 2 h daily to inhalants for 4 weeks via whole-body exposure chambers (inExpose system by SciReq). All mice were placed into the chambers for 2 h per day. CPC-APC mice were divided into four groups: a) Control, air-exposed, b) cig smoke-exposed (positive control), c) E.cig aerosol, commonly called E.cig vapor (EV), exposed, and d) Dual exposure to both cig and E.cig, to seek if co-use of both increases risk of CRC (Fig. 1A). Each exposure type had a dedicated chamber, tubing and pump, to prevent cross-contamination. For EV generation, the following vaping parameters were used: inhalation cycles per minute (Nc = 3), drag time (Dt = 4 s), peak inhalation flow (Qi = 2L/s), and electrical driving power (W = 6–8 watts based on the JUUL coil of 1.6 ohms and voltage of 3.7), using e-liquid composition of 30% propylene glycol (PG), 70% glycerin (Gly), and 59 mg/mL nicotine, which match the most popular e-liquids used by fourth-generation, pod-based e-devices (JUUL, FLUM, Sourin, etc.). For cigarette smoke generation, Nc = 1, Dt = 1 s, and Qi 2L/s were used, and 1R6F research cigarettes with the filter removed were replaced when < 1 cm remains (~ 1 cigarette every 8–10 min). For Dual exposures, mice were exposed to EV for 60 min and Cig for 60 min each day, yielding 2 h of inhalant exposure daily. Mice were exposed for 4 weeks, colons of mice were harvested, and the polyp number and area were measured.

Quantification of colon adenomas

Upon harvesting, each colon was cleaned and laid flat next to a ruler. Pictures of the colon were taken. Then, the pictures were uploaded into ImageJ software, which was used to count the number of polyps present in the mouse colons after exposure. The area of each polyp was also determined, and the combined total area of all polyps was calculated.

Histologic analysis of the colon

Colon tissues were fixed using 10% formalin overnight. Tissues were embedded in paraffin blocks, sectioned onto slides, and stained with H&E. The histologic score was determined by assessment of mucosal architecture and crypt loss and infiltration of immune cells in different colon regions (lamina propria, mucosa, and submucosa) as described previously (Erben et al. 2014).

Inflammatory and proliferation gene expression assessment by RT-qPCR

Total RNA was extracted from polyp regions (less than 25 mg) using the ZymoRNA miniprep kit (Zymo Research) and complementary DNA (cDNA) was reverse transcribed using qScript cDNA SuperMix (Quantabio). Real-time RT-PCR was performed using 2 × SYBR Green qPCR Master Mix (Biomake) and Bio-Rad CFX Opus 96 Real-Time PCR System (Bio-Rad). 18srRNA and GAPDH were used as housekeeping genes for normalization. The ΔΔCt method was used for the calculation as done previously (Schmittgen and Livak 2008). The relative fold change in the transcript level was calculated. The primer sequences used are listed in Table 1.

Quantification of DNA strand breaks by long amplicon (LA) qPCR

The detection of accumulated DNA strand breaks was performed as described previously (Sayed et al. 2020a). Briefly, genomic DNA was extracted from polyps (one polyp per mouse and 3 mice/each group of exposure), and levels of DNA strand breaks in polß (6.5 Kb) and globin (8.7 Kb) were quantified as described previously (Chakraborty et al. 2015). Briefly, the extracted DNA was amplified using the following condition: 94 °C for 30 s (94 °C for 30 s, 55–60 °C for 30 s depending on the oligo annealing temperature, and 65 °C for 10 min) for 25 cycles and 65 °C for 10 min. The sequences of oligo were described before (Sayed et al. 2020a; Chakraborty et al. 2015). Short amplicons for these genes were used to normalize the amplification of long amplicons.

Immunofluorescence for detection of DNA damage

Colon sections were deparaffinized in xylene, rehydrated in graded ethanol, and rinsed in Tris–phosphate-buffered saline (TBS). Heat-induced antigen retrieval was performed in a microwave for 30 min at 98 °C in 0.01 M citrate buffer (pH6). After cooling for 20 min, sections were incubated in 1% Triton X-100 for 30 min, blocked in TBST with 10% donkey serum for 1 h, and then incubated with primary rabbit monoclonal antibody Phospho-Histone H2A.X (Ser139), to detect γH2AX, overnight at 4 °C (1:250, Cell Signaling, #9718). After subsequent washes in TBST, slides were incubated with secondary Donkey anti-Rabbit IgG (H + L) Alexa fluor plus 488 (1:500, Invitrogen, A32790) for 1 h in the dark. Immunofluorescence (IF) images were taken with a 40X objective using a Leica SP8 II confocal laser scanning microscope.

Detection the level of DNA damage and DNA repair proteins by western analyses

Colon polyps (one polyp per mouse and 3 mice/group of exposure) were lysed using the RIPA buffer and the assessment of the level of base excision repair (BER) enzymes was performed as described before (Sayed et al. 2020a). Briefly, after separation of the proteins via acrylamide gels and transfer onto nitrocellulose membranes, membranes were blocked with 5% w/v skimmed milk and incubated with appropriate primary rabbit-derived antibodies (NEIL2, NTH1, and OGG1; in-house antibodies used in 1:500 dilutions). gH2AX and H2AX antibodies were purchased from Cell Signaling Technology (cat# 9718 and 7613, respectively). Histone deacetylase 2 (HDAC2; GeneTex, Irvine, CA, USA) was used as a loading control. After washing steps, the membranes were incubated with anti-isotype secondary antibody (GE Healthcare) conjugated with horseradish peroxidase in 5% skimmed milk at room temperature. All the western blot (WB) images were quantified using the ImageJ automated digitizing system based on three independent gel images (n = 3).

Quantification of proliferation

Colonic polyp regions (one polyp per mouse and 5 mice/ each group of exposure) and non-polyp regions were homogenized in phosphate-buffered saline (PBS). The protein concentrations were measured, and equal amounts of each sample (~ 10 ug) were used in the Mouse Ki-67 (Ki-67) ELISA Kit (MyBiosource, Catalogue # MBS1601117) according to the manufacturer’s instructions. The standard curve was developed using Ki67 standards, and the quantity of Ki67 in each sample was determined by measuring the absorbance at 450 nm.

Statistics

Analyses were conducted in Graphpad prism V10 (La Jolla, CA). Data are presented as mean ± SEM and analyzed using non-parametric Student’s t test and One-Way ANOVA as specified. Results are considered significant if the p value is less than 0.05.

ResultsE.cig and dual exposures increase adenomatous polyp formation

Previous studies have shown that cigarette smoke (cig) increases the risk of CRC and adenoma formation in the CPC-APC murine model by affecting gut microbiota and gut barrier integrity (Wang et al. 2021; Bai et al. 2022). Further, our previous study showed that E.cig vaping induces inflammation and gut leakiness in healthy gut and wild-type mice (Sharma et al. 2021). Here, we assessed the impact of E.cig vaping and Dual exposure to both E.cig and cig on CRC risk and promotion of adenoma formation using the CPC-APC mouse model (Hinoi et al. 2007) (Fig. 1A). As expected, the polyp number and area were increased in the CPC-APC mice after cigarette smoke exposure (Fig. 1B–D). Interestingly, we found that E.cig exposure significantly increased the polyp number and area in exposed mice compared to air-exposed controls (Fig. 1B–D). Regarding Dual exposure (E.cig + cig), polyp number and polyp area were also increased (Fig. 1B–D), and the number of polyps was significantly higher in Dual-exposed mice relative to E.cig-exposed mice (Fig. 1C).

E.cig and dual exposures increase inflammatory responses in the colon

Inflammation in the colon and tissue damage are linked to an increase risk of CRC (Zhou et al. 2023; Nardone et al. 2023). Therefore, we assessed if E.cig exposure promotes adenoma formation by affecting the inflammatory signaling pathways. To this end, colons of exposed mice were stained with H&E (Fig. 2A) and the pathology score was calculated (Fig. 2B). The pathology score is based on the degree of infiltration of immune cells in the colon and the architecture of colon mucosa (Erben et al. 2014). The pathology score was significantly higher in E.cig-exposed mice, cig-exposed mice, and Dual-exposed mice compared to air-exposed controls (Fig. 2B). We also assessed transcript levels of inflammatory cytokines TNF-α, IL-6, MCP-1, and CXCL-1 (IL-8 homolog) in the polyps. We selected these cytokines, since our previous studies showed that E.cig vaping increased inflammatory transcripts in the gut, particularly MCP-1, IL-6, and IL-8, leading to intestinal barrier defects (Sharma et al. 2021). Also, our previous study showed that cancer-associated microbes could promote CRC progression in the enteroid-derived monolayer isolated from the colon of APC-min mice by affecting CXCL-1/IL-8 cytokines (Sayed et al. 2020a). Cigarette, E.cig, and Dual exposures significantly increased transcript levels of TNF-α, CXCL-1, MCP-1, and IL-6 in the polyp regions compared to air-exposed controls (Fig. 2C–F). Interestingly, the levels of CXCL-1, MCP-1, and IL-6 and were significantly higher in the polyps of Dual-exposed mice compared to the polyps of E.cig-exposed mice (Fig. 2D–F).

E.cig and dual exposures mediate accumulation of mutations via induction of dsDNA breaks and suppression of host DNA glycosylases

Induction of DNA damage and failure to repair damage lead to the accumulation of mutations and genomic instability which promote cancer initiation and progression (Hanahan and Weinberg 2011). In vivo animals exposed to E.cig had toxic metabolites from nicotine which led to O(6)-methyl-2′-deoxyguanosine DNA adducts which increase mutagenesis in human lung and bladder epithelial cells (Lee et al. 2018). E.cig aerosols are genotoxic to human oral and lung epithelial cells by increasing oxidative DNA damage (Ganapathy et al. 2017). Therefore, we asked if E.cig exposure promotes adenoma formation in the CRC murine model via inducing dsDNA breaks and mutations. To this end, we assessed the level of γH2AX (a marker of DNA double-strand breaks) in the colon of exposed mice by IF and WB (Fig. 3A, B). Levels of γH2AX were higher in E.cig-exposed mice, cig-exposed mice, and Dual-exposed mice compared to air-exposed controls (Fig. 3A–C). The highest level of γH2AX was seen in Dual-exposed mice (Fig. 3A–C). Then, we tested the effect of exposures on host DNA repair proteins, more specifically DNA glycosylases (NEIL1, NEIL2, NTH1, and OGG1). Previous studies have shown that conventional smoking and E.cig mediate host DNA damage by suppressing DNA glycosylase expression (Ganapathy et al. 2017; Lee et al. 2018). We found that E.cig, cig, and Dual exposures reduce expression of all the tested DNA glycosylases (NEIL2, NTH1, and NEIL1) except OGG1 (Fig. 3D, E). Also, we quantified DNA strand breaks in Polβ and β-globin genes from polyps using LA-qPCR (Fig. 3F, G). We observed a significantly higher level of DNA strand-break accumulation in both the Polβ and β-globin genes following exposure to E.cig, cig, and Dual exposures compared to air-exposed controls (Fig. 3F, G). Collectively, E.cig exposure promoted polyp formation in CPC-APC mice by inducing DNA breaks, DNA mutations, and downregulating DNA repair proteins/ DNA glycosylases.

E.cig and dual exposures upregulate cancer stem cell markers and proliferation markers in the colon to initiate the cancer program

Previous studies have shown that cigarette smoke promotes proliferation and epithelial-mesenchymal transition (EMT), and increases the activity of cancer stem cells (CSCs) in the lung (Hirata et al. 2022), kidney (Qian et al. 2018), bladder (Sun et al. 2020), skin (Lin et al. 2021), liver (Xie et al. 2019), and pancreas (Nimmakayala et al. 2018). The CSCs are a subpopulation of tumor cells that promote cancer initiation, progression, metastasis, and resistance to chemotherapy and radiation (Liu et al. 2023). There are several markers for CSCs such as cluster of differentiation 44 (CD44) (Thapa and Wilson 2016), Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) (Ihemelandu et al. 2019), and Doublecortin-like kinase 1 (Dclk1) (Chandrakesan et al. 2017). Ki-67 is another marker that plays a crucial role in the maintenance of colon CSCs, and it is a marker of cell proliferation (Cidado et al. 2016). We focused on these four markers since previous studies showed that these markers were associated with adenoma formation and CRC in the APC mouse model (Chandrakesan et al. 2017; Abbasi et al. 2020; Wielenga et al. 1999; Yanai et al. 2017). We queried whether E.cig exposure affects the level of colon CSCs. To this end, we analyzed the transcript levels of CD44, Lgr-5, DCLK1, and Ki67 in the polyp regions across exposure groups (Fig. 4A). Cigarette smoke significantly upregulated the four CSC markers (~ 4–eightfold) compared to air-exposed controls (Fig. 4B–E). Likewise, E.cig induced expression of CD44, Lgr-5, and Ki67, but not DCLK1, in polyp regions (Fig. 4B–E). Dual exposure caused a significant stimulation in the transcriptome of the CSCs markers (Fig. 4B–E), and mRNA levels of CD44 and DCLK1 were significantly higher in Dual-exposed mice compared to E.cig-exposed mice (Fig. 4B, C). To confirm our findings, we assessed Ki67+ cells in polyp regions by measuring protein levels of Ki67 in whole-cell extracts by ELISA (Fig. 4F). First, we assessed the Ki67 in the non-involved region and polyp region from the same mouse. As expected, the level of Ki67 was significantly higher in the polyp regions compared to the non-involved regions as it is a marker of cell proliferation (Fig. 4F). Then, we compared the Ki67 protein level in all exposure groups of mice in the polyp region. We found that the level of Ki67 was significantly higher in E-cig, cig, and Dual-exposed mice compared to air-exposed controls (Fig. 4G).

Discussion

The role of tobacco and cigarette smoke in fueling CRCs has been established through robust epidemiologic studies with long-term follow-up and substantiated by mechanistic studies (Wang et al. 2021; Liang et al. 2009; Botteri et al. 2020; Bai et al. 2022). Since many young teenagers shifted from conventional smoking to E.cig or use both (dual), it is crucial to study the hazardous effects of E.cig and/or dual exposure on human health, more specifically on CRC. E.cig vaping mediates intestinal barrier defects by activating the inflammatory responses and affecting the gut epithelial tight junctions (Sharma et al. 2021). Although a leaky gut is a risk factor for CRC, there is no report on the direct effect of E.cig on the development of CRC.

To study the effect of E.cig on CRC development, we used an established CPC-APC murine model that develops adenomas/polyps in the colon/rectum, around 3 months of age (Hinoi et al. 2007; Drummond et al. 1996). This model was used by other researchers to assess the impact of cigarette smoke on CRC progression and study the mechanistic pathways of cigarette smoke-mediate CRC (Wang et al. 2021). In this study, we assessed the impact of E.cig vaping on CRC by several microscopic, molecular, and immunological approaches. Mice exposed to E.cig, cig, or both developed a significantly higher number of polyps and a higher polyp area compared to the air-exposed mice, suggesting that E.cig and cig can fuel CRC progression. Interestingly, mice exposed to both E.cig and cig (dual) had a significantly higher number of polyps compared to mice exposed to E.cig vapor alone, suggesting that administration of both E.cig and cig is more hazardous than vaping E.cig alone. Similarly, Hamon and colleagues showed that dual exposure had more damaging effect on lung epithelial cells and in the functions of phagocyte compared to the single exposure (Hamon et al. 2024).

Next, we studied the possible pathways through which E.cig mediates CRC progression. We focused on inflammatory pathways, host DNA damage and repair pathways, and stemness markers associated with cancer stem cells. Chronic inflammation in the gut is fueling CRC, and patients with inflammatory bowel diseases are at increased risk of CRC development (Nardone et al. 2023). Herein, we found that E.cig, cig, and dual exposure stimulated the inflammatory signaling pathways in the polyp regions of the colon as shown by an increase in the infiltration of immune cells, pathology score, and upregulated the transcript levels of inflammatory cytokines, such as IL-6, MCP-1, TNF-α, and Cxcl-1. The transcript levels were higher in dual-exposed mice than in E-cig-exposed mice. Likewise, dual exposure stimulated higher IL-8 than the single exposure using in vitro cell culture models (Hamon et al. 2024). These data suggest that E.cig could mediate CRC via activation of the inflammatory pathways.

Given the known effect of conventional cigarette in inducing DNA damage, we also assessed the impact of E.cig on host DNA damage and host DNA repair proteins, more specifically base excision repair pathways and DNA glycosylases. We focused on DNA glycosylases, especially NEIL-1 and NEIL-2 enzymes, since our previous studies showed that gut pathogens can stimulate DNA damage and promote gastrointestinal cancers by suppression of NEIL-2 at both transcriptional and translational levels (Sayed et al. 2020a, 2020b). Our data showed that E.cig, cig, and dual exposures induce DNA breaks, and cause specific DNA double-strand breaks. Besides E.cig, cig, and dual exposures suppress DNA repair enzymes such as NEIL-2, NEIL-1, and NTH1, but not OGG1. The previous effects result in the accumulation of mutations and excessive DNA damage leading to genome instability and cancer. Similar to the inflammatory pathway, the effect of dual exposures on DNA damage was higher than E.cig exposure. A previous study by Lee and colleagues showed that aldehydes present in smoking and/or E.cig reduced the expression of nucleotide excision repair (NER) and base excision repair (BER) enzymes, more specifically proteins XPC (associated with NER) and 8-oxo guanine DNA glycosylase (OGG1/2, associated with BER) that are playing significant roles in assessing DNA damage mediated by O6-methyl-deoxuguanosine and initiating DNA repair (Lee et al. 2018). The reduced expression or mutations of NEIL enzymes and OGG1 are associated with an increased risk of cancers (Krokan and Bjørås 2013; Shinmura et al. 2016; Zhai et al. 2008; Ali et al. 2015). In the study, E.cig vaping did not affect the level of OGG1 in the mice gut. The discrepancy between our data and the previous study could be due to the organ analyzed, and further studies need to confirm this point. Regarding NEIL1, we did not observe a significant difference among the three groups, E.cig, cig, and dual exposure. The findings indicate that there was no synergistic effect of dual exposure on the level of host NEIL1. Regarding NTH1, we found that cigarette smoking reduced the level of NTH1 in the colon, which is consistent with the earlier reported study in lungs (Deslee et al. 2010). However, the effect of E.cig and dual exposure on NTH1 has not been studied earlier in detail. We observed that the synergistic effect of dual exposure does not have significant impact on NTH1 level, and the cig exposure alone can reduce NTH1 more than dual exposure or E.cig alone. This may be attributed to some compensatory mechanisms. Increased NTH1 level can be a marker of CRC or to reduce the DNA damages associated with cancer (Limpose et al. 2018). Collectively, the observed lower level of NTH1 in cig-exposed compared to dual-exposed mice could be due to the following: (a) compensatory host mechanisms as the damage of DNA/tumorigenesis was higher in dual-exposed group compared to cig-exposed group..; (b) a hallmark of increased tumorigenesis in dual exposed compared to cig-exposed group..; (c) the components of dual exposure did not have a synergistic effect on NTH1. Further studies need to assess these points. Collectively, these findings indicate that E.cig can promote CRC through suppression of host BER pathways and inducing double-strand DNA breaks resulting in the accumulation of mutations and genomic instability.

We also investigated if E.cig vapor affects CSCs in the colon of exposed mice. CSCs play several roles in metastasis, angiogenesis, and EMT (Liu et al. 2023). We focused on specific markers of CSCs such as CD44, Lgr-5, and DCLk1. CD44 is a surface glycoprotein, in which its expression is controlled by β-catenin/Tcf-4 signaling pathway. The over-expression of CD44 is associated with the loss of the APC gene and it is an early marker in adenoma-CRC progression (Wielenga et al. 1999). Deletion of CD44 from APCMin/+ mice reduces intestinal tumorigenesis (Zeilstra et al. 2008). Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) is another biomarker of CSCs, and it is a receptor for WNT ligands such as R-spondin. LGR-5 positive cells act as CSCs and promote cancer propagation through clonal expansion (Yanai et al. 2017). The loss of the APC gene can stimulate CSCs and induce transformation in the LGR-5 positive cells to promote adenoma-to-carcinoma progression (Fu et al. 2019). Doublecortin-like kinase 1 (DCLK1) is a potential label for CRC CSCs. It is overexpressed in CSCs and maintains the self-renewal and NOTCH1 survival signaling of CSCs resulting in increasing the tumor colony in APCMin/+ mice (Chandrakesan et al. 2017). Ki67 controls the transcriptome changes and steps of tumorigenesis. APC mice lacking Ki67 do not develop tumors (Mrouj et al. 2021). We found that E.cig, cig, and dual exposures increase the gene expression of CD44, Ki67, and Lgr-5 transcripts. Not only the transcript level, but they also significantly increased Ki67 protein levels compared to air-exposed mice. For Dclk1, cig and dual exposures upregulate the transcript level, but E.cig was not. It is not clear why E.cig alone could not upregulate the Dclk1 level. Future studies need to verify this point. Collectively, our data showed that E.cig and other vaping exposures activate the markers of CSCs in the tumor regions to mediate tumor proliferation and expansion.

This study has some advantages and limitations. It is the first study that showed E.cig exposure could affect CRC progression and it shows possible pathways responsible for this progression such as activation of inflammatory signaling, inducing DNA damage and suppression of DNA repair proteins, and activation of cancer stem cells. The limitation of the study is the number of CPC-APC mice used in cig and dual exposure is not large enough. Future studies are required including a) a bigger cohort of the mice with different time points, b) other models of CRC mice, and c) other brands of cig and e-cig. However, our results consistently revealed that dual exposure poses a higher risk compared to single E-cig exposure. Here, we showed a selected number of pathways associated with CRC following exposure. Future studies are ongoing to understand the global pathways that link E.cig exposures with CRC development.

In conclusion, we reported here that E.cig could promote CRC in CPC-APC mice by stimulating inflammatory pathways, activation of DNA damage, and promoting the activity of cancer stem cells. It is crucial to alert the public about the possible hazardous effects of E.cig alone or in combination with the conventional smoking.

Acknowledgements

The authors are grateful to UMass-Lowell CoRE Research Facility (CRF) Cell Analysis & Imaging Lab for the confocal microscopy used in this study. The authors thank the histology core facility at UMASS Chan Medical School (Histospring.com) for staining γH2AX in tissue specimens. The authors also thank Joshua Alcan-tara for technical assistance in mice breeding and euthanasia. Diagrams were designed using free software https://www.biorender.com/.

Funding

SD was supported by UMASS Lowell intramural grant, NIH under Grant Nos. DK107585 and AG069689. SD and IMS have funding from the Leona M., and Harry B. Helmsley Charitable Trust. LCA was supported by the Tobacco-Related Disease Research Program (TRDRP), under Grant Nos. T32SR5359 and T30IP0965, the NIH NHLBI, under Grant Nos. K24HL155884, R01HL147326, and R01HL137052, and the Department of Veterans Affairs (VA), Merit Award numbers 1I01BX004767 and 1I01BX006447. TKH was supported by National Institute of Health under Grant Nos. 2R01 NS073976 and R56NS073976.

Conflict of interest The authors declare no competing interest.

Ethical approval The manuscript includes animal experiments. The experimental design was approved by the University of California San Diego Institutional Animal Care and Use Committee (IACUC) policies under the animal protocol number (S18086 and S16021). No human data in this study.

Data availability

The data that support the findings of this study are present in the main text. For further inquiries, please contact the corresponding authors.

ReferencesAbbasiN, LongT, LiY, YeeBA, ChoBS, HernandezJE, MaE, PatelPR, SahooD, SayedIM (2020) DDX5 promotes oncogene C3 and FABP1 expressions and drives intestinal inflammation and tumorigenesis. Life Sci Alliance. 10.26508/lsa.202000772AliK, MahjabeenI, SabirM, MehmoodH, KayaniMA (2015) OGG1 mutations and risk of female breast cancer: meta-analysis and experimental data. Dis Markers 2015:690878. 10.1155/2015/69087826089588 BaiX, WeiH, LiuW, CokerOO, GouH, LiuC, ZhaoL, LiC, ZhouY, WangG (2022) Cigarette smoke promotes colorectal cancer through modulation of gut microbiota and related metabolites. Gut 71:2439–2450. 10.1136/gutjnl-2021-32502135387878 BotteriE, BorroniE, SloanEK, BagnardiV, BosettiC, PeveriG, SantucciC, SpecchiaC, van den BrandtP, GallusS (2020) Smoking and colorectal cancer risk, overall and by molecular subtypes: a meta-analysis. Am J Gastroenterol 115:1940–1949. 10.14309/ajg.000000000000080332773458 BozierJ, ChiversEK, ChapmanDG, LarcombeAN, BastianN, Masso-SilvaJA, ByunMK, McDonaldCF, Alexander CrottyLE, WeenMP (2020) The evolving landscape of electronic cigarettes: a systematic review of recent evidence. Chest. 10.1016/j.chest.2019.12.042ChakrabortyA, WakamiyaM, Venkova-CanovaT, PanditaRK, Aguilera-AguirreL, SarkerAH, SinghDK, HosokiK, WoodTG, SharmaG (2015) Neil2-null mice accumulate oxidized DNA bases in the transcriptionally active sequences of the genome and are susceptible to innate inflammation. J Biol Chem 290:24636–24648. 10.1074/jbc.M115.65814626245904 ChandrakesanP, YaoJ, QuD, MayR, WeygantN, GeY, AliN, SurebanSM, GudeM, VegaK (2017) Dclk1, a tumor stem cell marker, regulates pro-survival signaling and self-renewal of intestinal tumor cells. Mol Cancer 16:30. 10.1186/s12943-017-0594-y28148261 CidadoJ, WongHY, RosenDM, Cimino-MathewsA, GarayJP, FesslerAG, RasheedZA, HicksJ, CochranRL, CroessmannS (2016) Ki-67 is required for maintenance of cancer stem cells but not cell proliferation. Oncotarget 7:6281–6293. 10.18632/oncotarget.705726823390 DesleeG, Adair-KirkTL, BetsuyakuT, WoodsJC, MooreCH, GieradaDS, ConradiSH, AtkinsonJJ, ToenniesHM, BattaileJT (2010) Cigarette smoke induces nucleic-acid oxidation in lung fibroblasts. Am J Respir Cell Mol Biol 43:576–584. 10.1165/rcmb.2009-0221OC20008282 DeyS, MaitiAK, HegdeML, HegdePM, BoldoghI, SarkarPS, Abdel-RahmanSZ, SarkerAH, HangB, XieJ (2012) Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2. DNA Repair (Amst) 11:570–578. 10.1016/j.dnarep.2012.03.00522497777 DiergaardeB, VrielingA, van KraatsAA, van MuijenGN, KokFJ, KampmanE (2003) Cigarette smoking and genetic alterations in sporadic colon carcinomas. Carcinogenesis 24:565–571. 10.1093/carcin/24.3.56512663519 DrummondF, SowdenJ, MorrisonK, EdwardsYH (1996) The caudal-type homeobox protein Cdx-2 binds to the colon promoter of the carbonic anhydrase 1 gene. Eur J Biochem 236:670–681. 10.1111/j.1432-1033.1996.t01-1-00670.x8612644 ErbenU, LoddenkemperC, DoerfelK, SpieckermannS, HallerD, HeimesaatMM, ZeitzM, SiegmundB, KühlAA (2014) A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol 7:4557–457625197329 FuT, CoulterS, YoshiharaE, OhTG, FangS, CayabyabF, ZhuQ, ZhangT, LeblancM, LiuS (2019) FXR regulates intestinal cancer stem cell proliferation. Cell 176:1098–1112.e1018. 10.1016/j.cell.2019.01.03630794774 GanapathyV, ManyangaJ, BrameL, McGuireD, SadhasivamB, FloydE, RubensteinDA, RamachandranI, WagenerT, QueimadoL (2017) Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage. PLoS ONE 12:e0177780. 10.1371/journal.pone.017778028542301 GentzkeAS, CreamerM, CullenKA, AmbroseBK, WillisG, JamalA, KingBA (2019) Vital signs: tobacco product use among middle and high school students—United States, 2011–2018. Morb Mortal Wkly Rep 68:157GrodenJ, ThliverisA, SamowitzW, CarlsonM, GelbertL, AlbertsenH, JoslynG, StevensJ, SpirioL, RobertsonM (1991) Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66:589–600. 10.1016/0092-8674(81)90021-01651174 HamonR, ThredgoldL, WijenayakaA, BastianNA, WeenMP (2024) Dual exposure to E-cigarette vapour and cigarette smoke results in poorer airway cell, monocyte, and macrophage function than single exposure. Int J Mol Sci. 10.3390/ijms25116071HanahanD, WeinbergRA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674. 10.1016/j.cell.2011.02.01321376230 HinoiT, AkyolA, TheisenBK, FergusonDO, GreensonJK, WilliamsBO, ChoKR, FearonER (2007) Mouse model of colonic adenoma-carcinoma progression based on somatic Apc inactivation. Cancer Res 67:9721–9730. 10.1158/0008-5472.Can-07-273517942902 HirataN, HorinouchiT, KandaY (2022) Effects of cigarette smoke extract derived from heated tobacco products on the proliferation of lung cancer stem cells. Toxicol Rep 9:1273–1280. 10.1016/j.toxrep.2022.06.00136518441 IhemelanduC, NaeemA, ParasidoE, BerryD, ChaldekasK, HarrisBT, RodriguezO, AlbaneseC (2019) Clinicopathologic and prognostic significance of LGR5, a cancer stem cell marker in patients with colorectal cancer. Colorectal Cancer. 10.2217/crc-2019-0009KingBA, JonesCM, BaldwinGT, BrissPA (2020) The EVALI and youth vaping epidemics - implications for public health. N Engl J Med 382:689–691. 10.1056/NEJMp191617131951683 KrokanHE, BjøråsM (2013) Base excision repair. Cold Spring Harb Perspect Biol 5:a012583. 10.1101/cshperspect.a01258323545420 LeeHW, ParkSH, WengMW, WangHT, HuangWC, LeporH, WuXR, ChenLC, TangMS (2018) E-cigarette smoke damages DNA and reduces repair activity in mouse lung, heart, and bladder as well as in human lung and bladder cells. Proc Natl Acad Sci U S A 115:E1560–e1569. 10.1073/pnas.171818511529378943 LiangPS, ChenTY, GiovannucciE (2009) Cigarette smoking and colorectal cancer incidence and mortality: systematic review and meta-analysis. Int J Cancer 124:2406–2415. 10.1002/ijc.2419119142968 LimposeKL, TregoKS, LiZ, LeungSW, SarkerAH, ShahJA, RamalingamSS, WernerEM, DynanWS, CooperPK (2018) Over-expression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer. Nucleic Acids Res 46:4515–4532. 10.1093/nar/gky16229522130 LinS, MeiW, LaiH, LiX, WengH, XiongJ, LinX, ZengT, ZhangQ, LiuX (2021) Cigarette smoking promotes keratinocyte malignancy via generation of cancer stem-like cells. J Cancer 12:1085–1093. 10.7150/jca.5074633442406 LiuQ, GuoZ, LiG, ZhangY, LiuX, LiB, WangJ, LiX (2023) Cancer stem cells and their niche in cancer progression and therapy. Cancer Cell Int 23:305. 10.1186/s12935-023-03130-238041196 LüchtenborgM, WeijenbergMP, KampmanE, van MuijenGN, RoemenGM, ZeegersMP, GoldbohmRA, van ‘t VeerP, de GoeijAF, van den BrandtPA, (2005) Cigarette smoking and colorectal cancer: APC mutations, hMLH1 expression, and GSTM1 and GSTT1 polymorphisms. Am J Epidemiol. 10.1093/aje/kwi114McCarthyM E-cigarettes are major threat to young people’s health, says US surgeon general. 2016.MeiraLB, BugniJM, GreenSL, LeeCW, PangB, BorenshteinD, RickmanBH, RogersAB, Moroski-ErkulCA, McFalineJL (2008) DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice. J Clin Invest 118:2516–2525. 10.1172/jci3507318521188 MirboloukM, CharkhchiP, KianoushS, UddinSI, OrimoloyeOA, JaberR, BhatnagarA, BenjaminEJ, HallME, DeFilippisAP (2018) Prevalence and distribution of e-cigarette use among US adults: behavioral risk factor surveillance system, 2016. Ann Intern Med 169:429–43830167658 MorinPJ, SparksAB, KorinekV, BarkerN, CleversH, VogelsteinB, KinzlerKW (1997) Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275:1787–1790. 10.1126/science.275.5307.17879065402 MroujK, Andrés-SánchezN, DubraG, SinghP, SobeckiM, ChaharD, Al GhoulE, AznarAB, PrietoS, PirotN (2021) Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis. Proc Natl Acad Sci U S A. 10.1073/pnas.2026507118NardoneOM, ZammarchiI, SantacroceG, GhoshS, IacucciM (2023) Inflammation-driven colorectal cancer associated with colitis: from pathogenesis to changing therapy. Cancers (Basel). 10.3390/cancers15082389NimmakayalaRK, SeshacharyuluP, LakshmananI, RachaganiS, ChughS, KarmakarS, RauthS, VengojiR, AtriP, TalmonGA (2018) Cigarette smoke induces stem cell features of pancreatic cancer cells via PAF1. Gastroenterology 155:892–908.e896. 10.1053/j.gastro.2018.05.04129864419 O’LoughlinJ, WellmanRJ, PotvinL (2016) Whither the e-cigarette? Int J Public Health 61:147–148. 10.1007/s00038-016-0800-526971510 PerezMF, Crotty AlexanderLE (2020) Why is vaping going up in flames? Ann Am Thorac Soc 17:545–54931944819 QianW, KongX, ZhangT, WangD, SongJ, LiY, LiX, GengH, MinJ, KongQ (2018) Cigarette smoke stimulates the stemness of renal cancer stem cells via Sonic Hedgehog pathway. Oncogenesis 7:24. 10.1038/s41389-018-0029-729540668 ReganAK, PromoffG, DubeSR, ArrazolaR (2013) Electronic nicotine delivery systems: adult use and awareness of the ‘e-cigarette’ in the USA. Tob Control 22:19–23. 10.1136/tobaccocontrol-2011-05004422034071 SalamancaJC, Meehan-AtrashJ, VreekeS, EscobedoJO, PeytonDH, StronginRM (2018) E-cigarettes can emit formaldehyde at high levels under conditions that have been reported to be non-averse to users. Sci Rep 8:1–629311619 SarebøM, SkjelbredCF, BreisteinR, LotheIM, HagenPC, BockG, HansteenIL, KureEH (2006) Association between cigarette smoking, APC mutations and the risk of developing sporadic colorectal adenomas and carcinomas. BMC Cancer 6:71. 10.1186/1471-2407-6-7116545110 SayedIM, ChakrabortyA, Abd El-HafeezAA, SharmaA, SahanAZ, HuangWJM, SahooD, GhoshP, HazraTK, DasS (2020a) The DNA glycosylase NEIL2 suppresses fusobacterium-infection-induced inflammation and DNA damage in colonic epithelial cells. Cells. 10.3390/cells9091980SayedIM, SahanAZ, VenkovaT, ChakrabortyA, MukhopadhyayD, BimczokD, BeswickEJ, ReyesVE, PinchukI, SahooD (2020b) Helicobacter pylori infection downregulates the DNA glycosylase NEIL2, resulting in increased genome damage and inflammation in gastric epithelial cells. J Biol Chem 295:11082–11098. 10.1074/jbc.RA119.00998132518160 SayedIM, Masso-SilvaJA, MittalA, PatelA, LinE, MoshenskyA, ShinJ, BojanowskiCM, DasS, AkuthotaP (2021a) Inflammatory phenotype modulation in the respiratory tract and systemic circulation of e-cigarette users: a pilot study. Am J Physiol Lung Cell Mol Physiol 321:L1134–l1146. 10.1152/ajplung.00363.202134704852 SayedIM, El-HafeezAAA, MaityPP, DasS, GhoshP (2021b) Modeling colorectal cancers using multidimensional organoids. Adv Cancer Res 151:345–383. 10.1016/bs.acr.2021.02.00534148617 SchmittgenTD, LivakKJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108. 10.1038/nprot.2008.7318546601 SharmaA, LeeJ, FonsecaAG, MoshenskyA, KothariT, SayedIM, IbeawuchiSR, PranadinataRF, EarJ, SahooD (2021) E-cigarettes compromise the gut barrier and trigger inflammation. iScience 24:102035. 10.1016/j.isci.2021.10203533537654 ShinbashiM, RubinBK (2020) Electronic cigarettes and e-cigarette/vaping product use associated lung injury (EVALI). Paediatr Respir Rev 36:87–91. 10.1016/j.prrv.2020.06.00332653465 ShinmuraK, KatoH, KawanishiY, IgarashiH, GotoM, TaoH, InoueY, NakamuraS, MisawaK, MinetaH (2016) Abnormal expressions of DNA glycosylase genes NEIL1, NEIL2, and NEIL3 are associated with somatic mutation loads in human cancer. Oxid Med Cell Longev 2016:1546392. 10.1155/2016/154639227042257 SiegelRL, WagleNS, CercekA, SmithRA, JemalA (2023) Colorectal cancer statistics, 2023. CA Cancer J Clin 73:233–254. 10.3322/caac.2177236856579 SunX, SongJ, LiE, GengH, LiY, YuD, ZhongC (2020) Cigarette smoke supports stemness and epithelial-mesenchymal transition in bladder cancer stem cells through SHH signaling. Int J Clin Exp Pathol 13:1333–134832661469 ThapaR, WilsonGD (2016) The importance of CD44 as a stem cell biomarker and therapeutic target in cancer. Stem Cells Int 2016:2087204. 10.1155/2016/208720427200096 TsoiKK, PauCY, WuWK, ChanFK, GriffithsS, SungJJ (2009) Cigarette smoking and the risk of colorectal cancer: a meta-analysis of prospective cohort studies. Clin Gastroenterol Hepatol 7:682–688. e5. 10.1016/j.cgh.2009.02.01619245853 WangH, ChenX, GaoQ, LiuK, BiG, DengJ, ZhangX (2021) Smoking induces the occurrence of colorectal cancer via changing the intestinal permeability. J Buon 26:1009–101534268966 WielengaVJ, SmitsR, KorinekV, SmitL, KielmanM, FoddeR, CleversH, PalsST (1999) Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway. Am J Pathol 154:515–523. 10.1016/s0002-9440(10)65297-210027409 XieC, ZhuJ, WangX, ChenJ, GengS, WuJ, ZhongC, LiX (2019) Tobacco smoke induced hepatic cancer stem cell-like properties through IL-33/p38 pathway. J Exp Clin Cancer Res 38:39. 10.1186/s13046-019-1052-zYanaiH, AtsumiN, TanakaT, NakamuraN, KomaiY, OmachiT, TanakaK, IshigakiK, SaigaK, OhsugiH (2017) Intestinal cancer stem cells marked by Bmi1 or Lgr5 expression contribute to tumor propagation via clonal expansion. Sci Rep 7:41838. 10.1038/srep4183828176811 YuV, RahimyM, KorrapatiA, XuanY, ZouAE, KrishnanAR, TsuiT, AguileraJA, AdvaniS, Crotty AlexanderLE (2016) Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines. Oral Oncol 52:58–65. 10.1016/j.oraloncology.2015.10.01826547127 ZeilstraJ, JoostenSP, DokterM, VerwielE, SpaargarenM, PalsST (2008) Deletion of the WNT target and cancer stem cell marker CD44 in Apc(Min/+) mice attenuates intestinal tumorigenesis. Cancer Res 68:3655–3661. 10.1158/0008-5472.Can-07-294018483247 ZhaiX, ZhaoH, LiuZ, WangLE, El-NaggarAK, SturgisEM, WeiQ (2008) Functional variants of the NEIL1 and NEIL2 genes and risk and progression of squamous cell carcinoma of the oral cavity and oropharynx. Clin Cancer Res 14:4345–4352. 10.1158/1078-0432.Ccr-07-528218594018 ZhangL, ShayJW (2017) Multiple roles of APC and its therapeutic implications in colorectal cancer. J Natl Cancer Inst. 10.1093/jnci/djw332ZhouRW, HarpazN, ItzkowitzSH, ParsonsRE (2023) Molecular mechanisms in colitis-associated colorectal cancer. Oncogenesis 12:48. 10.1038/s41389-023-00492-037884500 ZhuS-H, SunJY, BonnevieE, CumminsSE, GamstA, YinL, LeeM (2014) Four hundred and sixty brands of e-cigarettes and counting: implications for product regulation. Tob Control 23:3–9

E.cig and Dual exposures increase polyp formation in CRC-APC mice. CPC-APC mice (murine CRC model) were exposed to Air, E.cig, Cig, and E.cig + Cig (Dual exposure). Colonic tissue was collected after 4 weeks of exposure. A Experimental design infographic. B Representative mouse colon pictures from each exposure. C The number of polyps per mouse (n = 6–12 per group). D The sum of polyp diameter (mm) was calculated per mouse (n = 6–12 per group). Each dot represents one mouse. Data shown are means ± SEM. *P value < 0.05, **P value < 0.01, and ***P value < 0.001, as determined by the Mann–Whitney test. *indicates comparison to air-exposed controls; #indicates comparison to E.cig-exposed mice

E.cig and Dual exposures increase inflammatory responses in the colon. A Representative images of colons of CPC-APC mice from the four different conditions, stained with H&E. B Colon histology score was determined based on quantity of inflammatory cells in different regions (lamina propria, mucosa, and submucosa) and the morphology of mucosal architecture. C–F mRNA levels of inflammatory cytokines in colonic polyps, including TNF-α (C), CXCL-1 (D), MCP-1 (E), and IL-6 (F). Data presented as means ± SEM. *P value < 0.05, **P value < 0.01, and ***P value < 0.001, as determined by the Mann–Whitney test. *indicates comparison to air-exposed controls; #indicates comparison to E.cig-exposed mice, P < 0.05

E.cig and Dual exposures induce double-strand (ds) DNA breaks and suppress host DNA repair glycosylases. A Colonic polyps were stained for γH2AX for dsDNA breaks (green) and DAPI for nuclei staining (blue) and assessed with immunofluorescence (IF). Images were taken using 40X magnification, and the scale bar is 50 μm. B Immunoblot to γH2AX levels in the extract of polyps. Total H2AX was used for normalization. Three independent experiments (n = 3) were performed, and one representative figure is shown. C The band density of γH2AX normalized to total H2AX was calculated for each sample (analyzed with one-way ANOVA). D Immunoblot of NEIL2, NEIL-1, NTH1, and OGG1 levels in the extract of polyps. HDAC2 (histone deacetylase 2) was used as loading control. Three independent experiments (n = 3) were performed, and one representative figure is shown. E The band density of DNA glycosylases (NEIL-1, NEIL-2, NTH1, and OGG1) normalized to HDAC2 was calculated in each sample (analyzed with two-way ANOVA). F The polyps of exposed mice were harvested for genomic DNA isolation. Long amplicon quantitative PCR (LA-qPCR) was performed to evaluate the level of DNA strand-break accumulation. Representative gels show the amplification of each long fragment (~ 7–8 kB; upper panel) normalized to that of a short fragment (~ 250 bp; lower panel) of the corresponding (Pol β and β-Globin) genes. G The relative band density in (F) was calculated, and compared with air-exposed controls. Data presented as means ± SEM. *P value < 0.05, **P value < 0.01, and ***P value < 0.001. *indicates comparison to air-exposed controls; #indicates comparison to E.cig-exposed mice, P < 0.05; $ indicates comparison to cig-exposed mice, P < 0.05

E.cig and Dual exposure upregulates stemness and proliferation markers in the colon. A The experimental design showing analysis of the mRNA levels of cancer stem cell markers and EMT from the polyp of each exposed group. B–E The total RNA extracted from the polyp of each exposed group was assessed for CD-44 (B), DCLK-1 (C), ki67 (D), and Lgr-5 (E) by RT-qPCR. F The level of Ki67 in the whole polyp extract was measured by ELISA (n = 5 per each group). Each dot represents one mouse. Data presented as means ± SEM. *P value < 0.05, **P value < 0.01, and ***P value < 0.001, as determined by the Mann–Whitney test. *indicates comparison to air-exposed controls; #indicates comparison to E.cig-exposed mice, P < 0.05

Primer sequences for RT-qPCR

PrimersForward primer (3′–5′)Reverse primer (3′–5′)
18S rRNAGTAACCCGTTGAACCCCATTCCATCCAATCGGTAGTAGCG
Mouse GAPDHCATCACTGCCACCCAGAAGACTGATGCCAGTGAGCTTCCCGTTCAG
Mouse TNF-αCCACCACGCTCTTCTGTCTAAGGGTCTGGGCCATAGAACT
Mouse CXCl-1CTGGGATTCACCTCAAGAACATCCAGGGTCAAGGCAAGCCTC
Mouse MCP-1AAGTGCAGAGAGCCAGACGTCAGTGAGAGTTGGCTGGTG
Mouse IL-6CCCCAATTTCCAATGCTCTCCCGCACTAGGTTTGCCGAGTA
Mouse CD44CCTTGGCCACCACTCCTAATACTTGGATGGTTGTTGTGGGC
Mouse Lgr-5CCTACTCGAAGACTTACCCAGTGCATTGGGGTGAATGATAGCA
Mouse DCLK1CCTAAGACAGAACCCGCACCGCTAGGGAAGGCTAAGGCTG
Mouse ki67AGAGCTAACTTGCGCTGACTTCAATACTCCTTCCAAACAGGCA