In accordance with the well-accepted 3R strategy (the effort to “replace, reduce, refine” the use of animal models in research²), we have devised a series of human in vitro models which will avoid species translatability issues. These models are composed of human primary cells, cultured in two- or three-dimensions, which can be exposed to individual smoke constituents and fractions (eg, 2D cultures such as normal human bronchial epithelial cells or human endothelial cells) or to whole aerosols from Reduced-Risk Products (RRPs) or cigarette smoke (eg, 3D cultures).

By using systems toxicological risk assessment approaches, combining computable biological network models and gene expression changes, the molecular alterations triggered after different exposure conditions are analyzed and integrated with other biological endpoints. The measured biological alterations are also quantified, allowing the direct comparison of the impact triggered by exposure to cigarette smoke or RRP aerosols, thus providing evidence on the potential risk reduction.

Human 2D models and High Content Screening

High Content Screening (HCS) is an automated technology used to carry out functional / biological assays in a rapid, robust and cost-effective manner. The technology is based on the integration of three elements:

A state-of the art fluorescence microscope that allows the visualisation of cells and cellular structures.

A high-throughput image acquisition system that allows the automatic acquisition of hundreds of microscope images in a rapid and reproducible manner.

A software tool that enables the analysis and quantification of the biological signals in each one of the acquired images.

Using a combination of antibodies (capable of identifying specific components in the cell) and fluorescent dyes, the HCS technology can detect changes in target intensity, localisation and cellular morphology in live or fixed cell³.

At PMI, we use a large battery of HCS assays to investigate how specific substances alter the phenotype of a cell in a particular manner. Currently, we have established more than 10 different assays, allowing us to measure more than 15 different cellular parameters in cellular types representing the lung (normal human bronchial epithelial cells) and the cardiovascular system (human coronary artery endothelial cells)⁴.

Human 3D models

While 2D models have their uses, traditional 2D cell cultures in plastic dishes or flasks are limited in that the culture medium that covers them does not allow for direct exposure to complex aerosols. In addition, 2D cell cultures do not satisfactorily recapitulate the physiological cell-cell interaction conditions that cells experience in vivo.

To address these issues, in recent years researchers have looked for ways to culture cells in a more physiologically relevant manner. It has been demonstrated that by culturing normal human bronchial epithelial cells at the air-liquid interface, they are able to differentiate into a bronchial epithelium-like tissue with all the morphological properties that define the bronchial epithelium in vivo⁵. A 3D airway cellular model has also been built where cells express a comparable gene expression profile to tracheal and bronchial epithelial cells that have been collected in the clinical setting by brushing human airways⁶.

Cell culture in 3D has been touted as ‘biology's new dimension’⁷ and the National Institutes of Health, the US Food and Drug Administration, the Defense Advanced Research Projects Agency and the Defense Threat Reduction Agency are sponsoring a variety of projects focused on developing 3D organ models⁸.

At PMI we are using human 3D cultures that represent the different airway epithelia (nasal, oral, gingival and bronchial) that are directly exposed to cigarette smoke when inhaled by a smoker. These models are cultured with an air-liquid interface, allowing us to test whole cigarette smoke and RRP aerosols using the VITROCELL® system. We recently demonstrated that nasal, bronchial and oral in vitro systems are reliable models to assess RRPs by comparing their response to cigarette smoke exposure against in vivo tissue counterparts^9,10,11,12.

[1] National Research Council Committee on Toxicity Testing Assessment of Environmental Agents. Toxicity testing in the 21st century: a vision and a strategy. 2007: National Academies Press.

[2] Russell, WMS, RL Burch, and CW Hume. The principles of humane experimental technique. 1959.

[3] Thermo Scientific. CellInsight NXT High Content Screening Platform Brochure. 2012.

[4] Gonzalez-Suarez, I, et al. Systems biology approach for evaluating the biological impact of environmental toxicants in vitro. Chem Res Toxicol, 2014. 27(3): p. 367-76.

[5] Karp, PH, et al. An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures. Methods Mol Biol, 2002. 188: p. 115-37.

[6] Pezzulo, AA, et al. The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am J Physiol Lung Cell Mol Physiol, 2011. 300(1): p. L25-31.

[7] Abbott, A. Cell culture: Biology's new dimension. 2014.

[8] Baker, M. Tissue models: a living system on a chip. Nature, 2011. 471(7340): p. 661-5.

[9] Mathis, C, et al Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers. Am J Physiol Lung Cell Mol Physiol, 2013. 304(7): p. L489-503.

[10] Iskandar, AR, et al. Systems approaches evaluating the perturbation of xenobiotic metabolism in response to cigarette smoke exposure in nasal and bronchial tissues. Biomed Res Int, 2013. 2013: p. 512086.

[11] Talikka, M, et al. The response of human nasal and bronchial organotypic tissue cultures to repeated whole cigarette smoke exposure. Int J Toxicol, 2014.

[12] Schlage, WK, et al. In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures. Toxicol Mech Methods, 2014. 24(7): p. 470-87.

In Vivo Disease Models

In the assessment of our Reduced-Risk Products (RRPs) the key diseases of interest are cardiovascular disease, chronic obstructive pulmonary disease (COPD) and lung cancer, each of which has well established epidemiological links between their development and cigarette smoking.

It is well established that with ongoing use of cigarettes, the risk for these and other diseases increases over time. It is also known that when a smoker quits, and remains abstinent from cigarettes (smoking cessation), their risk of disease then decreases, potentially to same level of risk as someone who has never smoked. Our key aspiration with RRPs is to demonstrate that they have a risk reduction profile approaching that of cessation.

Cardiovascular disease

It has been shown that cigarette smoking aggravates and speeds up the growth of atherosclerosis plaques, leading to increased incidence of cardiovascular disease. Plaques may block the blood flow through the affected artery in organs such as the heart (where a heart attack can result), brain (which can lead to stroke) or legs (where blockage can lead to peripheral artery disease)⁸. The in vivo model we use for cardiovascular disease is the apolipoprotein E deficient (ApoE^-/-) mouse^9,2.

Chronic obstructive pulmonary disease (COPD)

Cigarette smoking is an important and significant risk factor for the development of COPD. The condition is associated with chronic inflammation, affecting mainly the lung parenchyma and the peripheral airways, and it is believed that oxidative stress caused by cigarette smoke exposure is the driving factor behind this^10,11. COPD can be considered to be a syndrome with two main pathologies, namely emphysema (lung tissue destruction) and chronic bronchitis. The in vivo models we use for COPD are the ApoE^-/- mouse² and C57BL/6 mouse^12,13. We have recently tested a prototype RRP in our in vivo model of COPD.

Lung cancer

Cigarette smoking is the largest and most significant cause of lung cancer, attributing to 80-90% of cases of the disease¹⁴. Lung cancers are classified according to their histological type, and can be broadly classified into one of two main types: non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). There are three main subtypes of NSCLC: adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma¹⁴. 40% of lung cancers due to smoking are adenocarcinomas, about 30% are squamous cell carcinomas and approximately 9% are large-cell carcinomas. The remainder of lung cancers caused by smoking are SCLCs, which are typically very aggressive⁷. The in vivo model we use for lung cancer is the AJ mouse³.

All our non-human in vivo research is restricted to those occasions where there are no alternatives available.

[1] Russell, WMS, RL Burch and CW Hume. The principles of humane experimental technique. 1959.

[2]Lietz, M, et al. Cigarette-smoke-induced atherogenic lipid profiles in plasma and vascular tissue of apolipoprotein E-deficient mice are attenuated by smoking cessation. Atherosclerosis, 2013. 229(1): p. 86-93.

[3] Stinn, W, et al. Lung inflammatory effects, tumorigenesis, and emphysema development in a long-term inhalation study with cigarette mainstream smoke in mice. Toxicol Sci, 2013. 131(2): p. 596-611.

[4] Xiang, Y, et al. Discovery of emphysema relevant molecular networks from an A/J mouse inhalation study using Reverse Engineering and Forward Simulation (REFS). Gene Regul Syst Bio, 2014. 8: p. 45-61.

[5] Phillips, BW, et al. A mechanistic study of cigarette smoke-induced COPD and cessation effects in C57BL/6 mice. International Conference on Toxicology. 2013: Seoul, South Korea.

[6] Boue, S. et al. Cigarette smoke induces molecular responses in respiratory tissues of ApoE(-/-) mice that are progressively deactivated upon cessation. Toxicology, 2013. 314(1): p. 112-24.

[7] Kogel, U, et al. A 28-day rat inhalation study with an integrated molecular toxicology endpoint demonstrates reduced exposure effects for a prototypic modified risk tobacco product compared with conventional cigarettes. Food Chem Toxicol, 2014. 68: p. 204-17.

[8] American Heart Association. Atherosclerosis. 2014.

[9] Boue, S, et al. Cigarette smoke induces molecular responses in respiratory tissues of ApoE(-/-) mice that are progressively deactivated upon cessation. Toxicology, 2013. 314(1): p. 112-24.

[10] Kirkham, PA and PJ Barnes. Oxidative stress in COPD. Chest, 2013. 144(1): p. 266-73.

[11] Rahman, I. The role of oxidative stress in the pathogenesis of COPD: implications for therapy. Treat Respir Med, 2005. 4(3): p. 175-200.

[12] Stinn, W, et al. Mechanisms involved in A/J mouse lung tumorigenesis induced by inhalation of an environmental tobacco smoke surrogate. Inhal Toxicol, 2005. 17(6): p. 263-76.

[13] Phillips, BW, et al. A mechanistic study of cigarette smoke-induced COPD and cessation effects in C57BL/6 mice, in International Conference on Toxicology. 2013: Seoul, South Korea.

[14] Horn, L, W Pao and DH Johnson. Chapter 89, in Harrison's principles of internal medicine, DL Longo, et al. Editors. 2012, McGraw-Hill.

These studies are designed alongside the studies carried out in our in vivo models of disease. Although many more experimental manipulations can be carried out in rodent models as compared to humans, it is generally acknowledged that there is no model that represents the totality of disease as it manifests in humans, especially for smoking-related diseases. Therefore the combination of experimental manipulation of rodent models, combined with physiological and molecular measurements in human subjects, allows us to build a much more comprehensive understanding of the mechanisms underlying smoking induced incidence of diseases such as cardiovascular disease, COPD and lung cancer.

Diseases associated with the smoking of cigarettes normally take 25-30 years to develop. As such, the standard way to assess if switching to RRPs lowers the risk of disease incidence would require 25-30 years of epidemiology data, post-launch of a new product, in order to determine if there is a reduction in risk. In order to accumulate clinical scientific evidence in a significantly shorter timeframe, we use the translational systems approach to identify molecular or physiological markers which may give us an indication of a reduction in disease in a much shorter timeframe.

Biomarkers of Disease Onset

A large amount of research is being conducted by the scientific community in the area of biomarker discovery for all three diseases of interest (cardiovascular disease, COPD and lung cancer). However, much of this research is aimed at identifying biomarkers of disease stage, or at identifying biomarkers indicating the efficacy of a therapeutic intervention.

One example is in the field of COPD biomarker discovery. The severity of COPD is defined by the Global Strategy for the Diagnosis, Management and Prevention of COPD (also known as GOLD) criteria, and ranges from stage 1 (mild) to stage 4 (very severe). All of the therapeutic options for COPD aim at stopping the progression of disease from one stage to the next, thus the biomarkers of interest are those that indicate the stage of disease a patient is currently in, and those that are associated with later stages of COPD. The figure below shows a graphical representation of this:

COPD Biomarkers

In addition to these biomarkers, we are also interested in identifying biomarkers which precede the clinical onset of the disease and are associated with the exposure to cigarette smoke. We have termed these biomarkers “Biomarkers of Disease Onset”.

The ultimate challenge with Biomarkers of Disease Onset is to show that they are mechanistically linked to the development of a specific disease and are indeed biomarkers which indicate the future development of disease.

Identifying Biomarkers of Disease Onset for COPD

The first disease that we are working on for the identification of Biomarkers of Disease Onset is COPD and we have recently conducted a study in this area. The study was designed alongside our in vivo COPD studies to ensure that we gained a comprehensive understanding of the mechanisms linking cigarette smoke exposure to COPD development. An overview of the study design is available on ClinicalTrials.gov and is summarised below.

Smoke / aerosol fractions and in vitro exposure

The classical way of exposing in vitro test systems to smoke or aerosol is to separate them into their separate phases. Aerosols from RRPs and smoke from cigarettes are made up of particles (the Total Particulate Matter, or TPM) and a mixture of volatile or vapour components (the Gas-Vapour Phase, or GVP). By passing the smoke or aerosol through a Cambridge glass fibre filter pad and a solution of phosphate buffered saline (PBS), separation of the TPM and GVP can be achieved.

The diagram below shows how the smoke / aerosol fractions are collected. The TPM is captured on a Cambridge filter pad, while GVP is the fraction of the smoke / aerosol not retained by the filter pad and soluble in PBS.

Use of the Vitrocell® 24/48 exposure system

As shown here, the system was used to determine levels of nicotine and eight carbonyls in whole cigarette smoke.

Nicotine / carbonyl levels in whole smoke from cigarettes.

[1] Thorne, D and J Adamson. A review of in vitro cigarette smoke exposure systems. Exp Toxicol Pathol, 2013. 65(7-8): p. 1183-93.

[2] Characterization of the Vitrocell(R) 24/48 aerosol exposure system using mainstream cigarette smoke. Chem Cent J, 2014. 8(1): p. 62.

[3] Adamson, J, et al. Assessment of an in vitro whole cigarette smoke exposure system: The Borgwaldt RM20S 8-syringe smoking machine. Chem Cent J, 2011. 5: p. 50.

[4] Scian, MJ, et al. Characterization of a whole smoke in vitro exposure system (Burghart Mimic Smoker-01). Inhal Toxicol, 2009. 21(3): p. 234-43.

[5] Scian, MJ, et al. Chemical analysis of cigarette smoke particulate generated in the MSB-01 in vitro whole smoke exposure system. Inhal Toxicol, 2009. 21(12): p. 1040-52.

[6] Adamson, J, et al. Assessment of cigarette smoke particle deposition within the Vitrocell(R) exposure module using quartz crystal microbalances. Chem Cent J, 2013. 7: p. 50.

[7] Phillips, J, et al. Exposure of bronchial epithelial cells to whole cigarette smoke: assessment of cellular responses. Altern Lab Anim, 2005. 33(3): p. 239-48.

[8] Schlage WK et al Analytical in vitro approach for studying cyto- and genotoxic effects of particulate airborne material. Anal Bioanal Chem, 2011. 401(10): p. 3213-20.

[9] Rach, J, et al. Direct exposure at the air-liquid interface: evaluation of an in vitro approach for simulating inhalation of airborne substances. J Appl Toxicol, 2014. 34(5): p. 506-15.