Summary of Study ST002823

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001765. The data can be accessed directly via it's Project DOI: 10.21228/M8K128 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002823
Study Title	Smoking Induced Gut Microbial Dysbiosis Mediates Cancer Progression Through Adaptive Immune System Modulation
Study Summary	Cigarette smoke exposure (CSE), either through active smoking or secondhand smoke, increases the risk for a plethora of cancers. Studies have estimated that one in three cancer deaths is associated with cigarette smoke exposure. However, despite ongoing research on numerous carcinogens, the underlying mechanism(s) remain poorly understood. Recent evidence indicates that the gut microbiome can influence cancer progression by immune system modulation. Since CSE alters the gut microbiome, we hypothesized that the gut microbiome serves as a causative link between smoking and cancer growth. Through a combination of rigorous syngeneic animal models and fecal microbiome transplantation studies, we establish an essential role for smoke-induced dysbiosis in cancer growth. Using Flow cytometric analysis of tumor specimens and experiments in Rag 1 KO and CD8 KO, we demonstrate that smoke induced tumor growth requires functional adaptive immunity. We further characterized the unique gut microbial and metabolomic signatures induced by CSE using high throughput 16s rRNA sequencing and mass spectrometric techniques. Finally, utilizing gut microbial ablation strategies with broad and narrow-spectrum antibiotics, we demonstrate the reversal of phenotypic effects of CSE and present a novel actionable target to mitigate CSE-induced tumor promotion.
Institute	University of Alabama, Birmingham
Department	Department of Surgery
Last Name	Vikas
First Name	Dudeja
Address	1808 7th Avenue South Boshell Building- Suite 573 Birmingham, AL 35294
Email	vdudeja@uabmc.edu
Phone	205 975 7836
Submit Date	2023-08-18
Raw Data Available	Yes
Raw Data File Type(s)	wiff
Analysis Type Detail	LC-MS
Release Date	2025-02-21
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001765
Project DOI:	doi: 10.21228/M8K128
Project Title:	Smoking Induced Gut Microbial Dysbiosis Mediates Cancer Progression Through Adaptive Immune System Modulation
Project Summary:	Cigarette smoke exposure (CSE), either through active smoking or secondhand smoke, increases the risk for a plethora of cancers. Studies have estimated that one in three cancer deaths is associated with cigarette smoke exposure. However, despite ongoing research on numerous carcinogens, the underlying mechanism(s) remain poorly understood. Recent evidence indicates that the gut microbiome can influence cancer progression by immune system modulation. Since CSE alters the gut microbiome, we hypothesized that the gut microbiome serves as a causative link between smoking and cancer growth. Through a combination of rigorous syngeneic animal models and fecal microbiome transplantation studies, we establish an essential role for smoke-induced dysbiosis in cancer growth. Using Flow cytometric analysis of tumor specimens and experiments in Rag 1 KO and CD8 KO, we demonstrate that smoke induced tumor growth requires functional adaptive immunity. We further characterized the unique gut microbial and metabolomic signatures induced by CSE using high throughput 16s rRNA sequencing and mass spectrometric techniques. Finally, utilizing gut microbial ablation strategies with broad and narrow-spectrum antibiotics, we demonstrate the reversal of phenotypic effects of CSE and present a novel actionable target to mitigate CSE-induced tumor promotion.
Institute:	University of Alabama, Birmingham
Last Name:	Vikas
First Name:	Dudeja
Address:	1808 7th Avenue South Boshell Building- Suite 573 Birmingham, AL 35294
Email:	vdudeja@uabmc.edu
Phone:	205 975 7836

Subject:

Subject ID:	SU002932
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammals

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Factors
SA305591	12-5-2020Ctr9- Fecal	Control
SA305592	12-5-2020 Ctrl10- Fecal	Control
SA305593	12-5-2020ctrl7- Fecal	Control
SA305594	12-5-2020Ctrl1- Fecal	Control
SA305595	12-5-2020Ctrl8- Fecal	Control
SA305596	12-5-2020Ctrl6- Fecal	Control
SA305597	12-5-2020Ctrl2- Fecal	Control
SA305598	12-5-2020Ctrl4- Fecal	Control
SA305599	12-5-2020Ctrl3- Fecal	Control
SA305600	12-5-2020Ctrl5- Fecal	Control
SA305601	12-5-2020Smoke 8- Fecal	Smoke
SA305602	12-5-2020Smoke 7- Fecal	Smoke
SA305603	12-5-2020Smoke 10- Fecal	Smoke
SA305604	12-5-2020Smoke11- Fecal	Smoke
SA305605	12-5-2020Smoke 9- Fecal	Smoke
SA305606	12-5-2020Smoke5- Fecal	Smoke
SA305607	12-5-2020Smoke 6- Fecal	Smoke
SA305608	12-5-2020Smoke 1- Fecal	Smoke
SA305609	12-5-2020Smoke 2- Fecal	Smoke
SA305610	12-5-2020Smoke 4- Fecal	Smoke
SA305611	12-5-2020Smoke3- Fecal	Smoke
SA305612	12-5-2020Smoke+Neo7- Fecal	Smoke+ Neo
SA305613	12-5-2020Smoke+ neo8- Fecal	Smoke+ Neo
SA305614	12-5-2020Smoke+ neo6- Fecal	Smoke+ Neo
SA305615	12-5-2020Smoke+Neo3- Fecal	Smoke+ Neo
SA305616	12-5-2020Smoke+ Neo1- Fecal	Smoke+ Neo
SA305617	12-5-2020smoke+ Neo2- Fecal	Smoke+ Neo
SA305618	12-5-2020Smoke+Neo4- Fecal	Smoke+ Neo
SA305619	12-5-2020Smoke+Neo5- Fecal	Smoke+ Neo

Showing results 1 to 29 of 29

Showing results 1 to 29 of 29

Collection:

Collection ID:	CO002925
Collection Summary:	Animal models All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines. C57BL/6J WT mice, A/J mice (stock no. 000646), Rag1 KO mice (B6.129S7-Rag1tm1Mom/J), as well as CD8 KO mice (B6.129S2-Cd8atm1Mak/J), were purchased from the Jackson Laboratory (Bar Harbor, ME). LSL- KrasG12D Pdx-1cre (KC) mice were generated in our breeding facility by crossing LSL-KrasG12D mice with Pdx-1cre mice. Mice were backcrossed to the C57BL/6J genetic background for at least ten generations. Experiments with WT, Rag1 KO, and CD8 KO were performed with only female mice 6-8 weeks old. Mice of both sexes were recruited for KC mice experiments. Littermates of genetically modified mice were used as controls. Animal experiments were authorized and overseen by the Institutional Animal Care and Use Committee (IACUC) in accordance with approved protocols. Smoke chamber The TE-10 smoking machine (Teague Enterprises) was used to provide smoke exposure to mice. Humidified 3R4F cigarettes (Tobacco Health Research Institute, Lexington, KY) were used to provide cigarette smoke. A smoke concentration of 150-200 mg/mm3 was maintained inside the chamber, and carbon monoxide levels were monitored. Mice were acclimated to cigarette smoke during the first week through incremental increases in smoke exposure from 1 hour/day to 4 hours/day. This level of exposure was continued for the duration of the experiment. Mice kept in a compartment with room air flow instead of cigarette smoke were used as controls.
Sample Type:	Feces

Treatment:

Treatment ID:	TR002941
Treatment Summary:	In vivo experiments For smoke chamber experiments with WT mice, CD8 KO mice, or Rag1 KO mice, 6–8-week-old female mice were utilized. For antibiotic depletion experiments (using WT mice or Rag1 KO mice), mice were divided into four conditions - control mice, mice with smoke exposure alone, mice receiving antibiotics alone, and mice receiving smoke exposure along with antibiotics. The pre-exposure phase was four weeks, during which mice were given cigarette smoke and/or antibiotics. Subsequently, the mice were challenged with subcutaneous tumors. We first standardized the pre-exposure and found that four weeks was sufficient time to develop the tumor-promoting effects of smoke. The broad-spectrum antibiotics included - vancomycin (100 mg/kg), ampicillin (200 mg/kg), metronidazole (200 mg/kg), neomycin (200 mg/kg), and amphotericin (1 mg/kg). Ampicillin was dissolved in drinking water, while the rest were dissolved in phosphate buffered saline (PBS) and administered as a daily gavage of 0.5 ml solution. Smoke and antibiotics were continued after the tumor challenge until the experimental endpoint. For the CD8 KO mice experiment, WT and CD8 KO mice were divided into two groups each - control mice and smoke-exposed mice. The smoke pre-exposure period lasted four weeks, after which mice were implanted with subcutaneous tumors. Exposure was continued until the endpoint. To test the effects of antibiotics in a therapeutic setting, WT mice were initially divided into control mice and smoke-exposed mice. After the pre-exposure phase, mice were challenged with subcutaneous tumors, which were allowed to grow for two weeks. Both groups were further randomized into two groups - one without an antibiotic cocktail and one with an antibiotic cocktail. For NNK experiments, a setup similar to the smoke chamber was used. Four groups were as followed - control mice, NNK alone, antibiotics alone, and NNK and antibiotics. NNK pre-exposure was given for six weeks through weekly injections, i.p 100 mg/kg. This was followed by a subcutaneous tumor challenge, and injections of NNK were continued until the endpoint. For the KC mice experiment, 8-week-old KC mice were randomly recruited to each of the four groups mentioned above. Weekly NNK injections and/or antibiotic cocktails were given for eight weeks, after which mice were euthanized at four months of age. A/J mice were given weekly NNK injections from 6-10 weeks of age, euthanized at 28 weeks, and individual tumor nodules in the lungs were recorded. For FMT experiments, stool was collected from cancer-naive control or smoke-exposed mice (4 weeks). The pellets were homogenized and dissolved in PBS. Eight to ten pellets were dissolved in 10 ml PBS. The mixture was then centrifuged at 500g for 5 minutes to allow fecal matter to settle down. The supernatant was collected and administered to recipient mice through oral gavage (500uL). The recipient mice were prepared for FMT by ablating their gut microbiome using a broad-spectrum antibiotic cocktail, as already described, for two weeks. A washout period of one week was allowed for the antibiotics to flush out of the system. This was followed by biweekly oral gavage of fecal microbiome from control or smoke-exposed mice for four weeks to reconstitute the microbiome. Subcutaneous tumors were then implanted, and biweekly FMT continued until the endpoint.

Treatment ID:

TR002941

Treatment Summary:

In vivo experiments For smoke chamber experiments with WT mice, CD8 KO mice, or Rag1 KO mice, 6–8-week-old female mice were utilized. For antibiotic depletion experiments (using WT mice or Rag1 KO mice), mice were divided into four conditions - control mice, mice with smoke exposure alone, mice receiving antibiotics alone, and mice receiving smoke exposure along with antibiotics. The pre-exposure phase was four weeks, during which mice were given cigarette smoke and/or antibiotics. Subsequently, the mice were challenged with subcutaneous tumors. We first standardized the pre-exposure and found that four weeks was sufficient time to develop the tumor-promoting effects of smoke. The broad-spectrum antibiotics included - vancomycin (100 mg/kg), ampicillin (200 mg/kg), metronidazole (200 mg/kg), neomycin (200 mg/kg), and amphotericin (1 mg/kg). Ampicillin was dissolved in drinking water, while the rest were dissolved in phosphate buffered saline (PBS) and administered as a daily gavage of 0.5 ml solution. Smoke and antibiotics were continued after the tumor challenge until the experimental endpoint. For the CD8 KO mice experiment, WT and CD8 KO mice were divided into two groups each - control mice and smoke-exposed mice. The smoke pre-exposure period lasted four weeks, after which mice were implanted with subcutaneous tumors. Exposure was continued until the endpoint. To test the effects of antibiotics in a therapeutic setting, WT mice were initially divided into control mice and smoke-exposed mice. After the pre-exposure phase, mice were challenged with subcutaneous tumors, which were allowed to grow for two weeks. Both groups were further randomized into two groups - one without an antibiotic cocktail and one with an antibiotic cocktail. For NNK experiments, a setup similar to the smoke chamber was used. Four groups were as followed - control mice, NNK alone, antibiotics alone, and NNK and antibiotics. NNK pre-exposure was given for six weeks through weekly injections, i.p 100 mg/kg. This was followed by a subcutaneous tumor challenge, and injections of NNK were continued until the endpoint. For the KC mice experiment, 8-week-old KC mice were randomly recruited to each of the four groups mentioned above. Weekly NNK injections and/or antibiotic cocktails were given for eight weeks, after which mice were euthanized at four months of age. A/J mice were given weekly NNK injections from 6-10 weeks of age, euthanized at 28 weeks, and individual tumor nodules in the lungs were recorded. For FMT experiments, stool was collected from cancer-naive control or smoke-exposed mice (4 weeks). The pellets were homogenized and dissolved in PBS. Eight to ten pellets were dissolved in 10 ml PBS. The mixture was then centrifuged at 500g for 5 minutes to allow fecal matter to settle down. The supernatant was collected and administered to recipient mice through oral gavage (500uL). The recipient mice were prepared for FMT by ablating their gut microbiome using a broad-spectrum antibiotic cocktail, as already described, for two weeks. A washout period of one week was allowed for the antibiotics to flush out of the system. This was followed by biweekly oral gavage of fecal microbiome from control or smoke-exposed mice for four weeks to reconstitute the microbiome. Subcutaneous tumors were then implanted, and biweekly FMT continued until the endpoint.

Sample Preparation:

Sampleprep ID:	SP002938
Sampleprep Summary:	Metabolites were extracted from fecal, and mouse liver pool was used as a quality control and followed the extraction procedure described. Briefly, 10 mg of tissue was used for the metabolic extraction. The extraction step starts with addition of 750μL ice-cold methanol: water (4:1) containing 20μL spiked internal standards (ISTDs). After homogenization, ice-cold chloroform and water were added in a 3:1 ratio for a final proportion of 4:3:2 methanol:chloroform:water. The organic and aqueous layers were collected, dried, and resuspended in methanol: water (1:1). The extract was deproteinized using a 3 kDa molecular filter and the filtrate was dried under vacuum. The dried extracts were re-suspended in 100 μL of injection solvent composed of 1:1 methanol: water and subjected to LC-MS.

Sampleprep ID:

SP002938

Sampleprep Summary:

Metabolites were extracted from fecal, and mouse liver pool was used as a quality control and followed the extraction procedure described. Briefly, 10 mg of tissue was used for the metabolic extraction. The extraction step starts with addition of 750μL ice-cold methanol: water (4:1) containing 20μL spiked internal standards (ISTDs). After homogenization, ice-cold chloroform and water were added in a 3:1 ratio for a final proportion of 4:3:2 methanol:chloroform:water. The organic and aqueous layers were collected, dried, and resuspended in methanol: water (1:1). The extract was deproteinized using a 3 kDa molecular filter and the filtrate was dried under vacuum. The dried extracts were re-suspended in 100 μL of injection solvent composed of 1:1 methanol: water and subjected to LC-MS.

Combined analysis:

Analysis ID	AN004603	AN004604
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	ABSciex 5600 Triple TOF	ABSciex 5600 Triple TOF
Column	Waters XBridge Amide (100 x 4.6mm,3.5um)	Waters XBridge Amide (100 x 4.6mm,3.5um)
MS Type	ESI	ESI
MS instrument type	Triple TOF	Triple TOF
MS instrument name	ABI Sciex 5600 TripleTOF	ABI Sciex 5600 TripleTOF
Ion Mode	POSITIVE	NEGATIVE
Units	area	area

Chromatography:

Chromatography ID:	CH003463
Chromatography Summary:	HILIIC-Positive
Instrument Name:	ABSciex 5600 Triple TOF
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Column Temperature:	37
Flow Gradient:	Gradient flow: 0-3 min 85% B; 3-12 min 30% B, 12-15 min 2% B, 16 min 95% B, followed by re-equilibration till the end of the gradient 23 min to the initial starting condition of 85% B.
Flow Rate:	0.4 mL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	HILIC

Chromatography ID:	CH003464
Chromatography Summary:	HILIIC-negative
Instrument Name:	ABSciex 5600 Triple TOF
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Column Temperature:	37
Flow Gradient:	0-3 min 85% B, 3-12 min 30% B, 12-15 min 2% B, 15-16 min 85% B followed by re-equilibration till the end of the gradient 23 min to the initial starting condition of 85% B.
Flow Rate:	0.4 mL/min
Solvent A:	100% water; 20 mM ammonium acetate, pH 9.0
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS004349
Analysis ID:	AN004603
Instrument Name:	ABI Sciex 5600 TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	For metabolomics, 5μL of the metabolite extract was injected into a 3.5μm particle 4.6×150mm X bridge amide column which heats to 60°C. 0.1% formic acid in water as solvent-A and acetonitrile as solvent-B in positive ionization and 20mM ammonium acetate in 95% water; 5% acetonitrile and 100% acetonitrile as solvent-B in negative ionization mode. The flow rate used for these experiments was 0.4 mL/min. The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source. The instrument performed one TOF MS survey scan (150ms) and 15 MS/MS scans with a total duty cycle time of 2.4s. The mass range in both modes was 50–1200 m/z. We controlled the acquisition in both MS and MS/MS spectra by data-dependent acquisition function of the Analyst TF software (AB Sciex, Concord, Canada). Rolling collision energy spread was set whereby the software calculated the collision energy value to be applied as a function of m/z. Mass accuracy was maintained by the use of an automated calibrant delivery system interfaced to the second inlet of the Duo Spray source.
Ion Mode:	POSITIVE

MS ID:	MS004350
Analysis ID:	AN004604
Instrument Name:	ABI Sciex 5600 TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	For metabolomics, 5μL of the metabolite extract was injected into a 3.5μm particle 4.6×150mm X bridge amide column which heats to 60°C. 0.1% formic acid in water as solvent-A and acetonitrile as solvent-B in positive ionization and 20mM ammonium acetate in 95% water; 5% acetonitrile and 100% acetonitrile as solvent-B in negative ionization mode. The flow rate used for these experiments was 0.4 mL/min. The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source. The instrument performed one TOF MS survey scan (150ms) and 15 MS/MS scans with a total duty cycle time of 2.4s. The mass range in both modes was 50–1200 m/z. We controlled the acquisition in both MS and MS/MS spectra by data-dependent acquisition function of the Analyst TF software (AB Sciex, Concord, Canada). Rolling collision energy spread was set whereby the software calculated the collision energy value to be applied as a function of m/z. Mass accuracy was maintained by the use of an automated calibrant delivery system interfaced to the second inlet of the Duo Spray source.
Ion Mode:	NEGATIVE