Complex 3D Human Tissue Cultures by Schauermann Tamás

COMPLEX 3D HUMAN TISSUE CULTURES The Way Forward Towards More Predictive Pre-Clinical Testing Using a 3D lung tissue system for disease modelling and to assess drug safety and efficacy

Finding a pre-clinical disease model, which predicts the efficacy and toxicity of any given drug candidate in humans is one of the greatest challenges of the pharmaceutical industry. The vast majority of lung toxicity and disease studies still rely upon 2D cell culture assays and animal models, but most of these techniques are simply unable to imitate the human in vivo environment, thus do not accurately predict the effects of the examined compound. There are also the unavoidable ethical issues associated with animal experiments. A 3D in vitro lung model system made from differentiated primary human lung cell types provided by Humeltis Laboratory might help to overcome these hurdles.

The lung is a complex organ, both anatomically and physiologically, characterised by a compartmentalised construction. Given the lung’s intricate structure, lung diseases often result in complex changes to its architecture and function, which can make the development of various treatment options quite challenging. It is therefore imperative that models used in the assessment of drug responses and disease pathology provide an accurate representation of the lung’s innately intricate structure and dynamic nature.

Modelling the lung – normal and diseased lung tissues as 3D cell cultures

With regulatory bodies such as the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) pushing the 3Rs (Replacement, Reduction and Refinement) there is mounting pressure to minimise the use of animal models in drug testing and to move towards in vitro models. Accurately modelling the lung in vitro during preclinical research stages is an important task from an ethical, financial and regulatory standpoint. Modelling lung diseases can help to define a molecular basis for pulmonary diseases and improve target identification and validation, leading to the development of a more effective drug. Recognising the pressing need to understand the extremely complex environment of the human lung, the Humeltis team have developed an in vitro lung tissue system using primary lung mesenchymal and epithelial cells. These constituents are combined in appropriate proportions and incubated together to form an accurate and functional representational of in vivo lung tissues. The primary cells used are from different parts of the lung – small airway, vascular and bronchial – to construct an accurate recreation of complex lung tissues. The cell types used include epithelial cells, endothelial cells, fibroblasts and peripheral blood monocytes (which differentiate into macrophages in the model). The lung tissue models uses differentiated primary lung cells in favour of stems cells to speed up tissue formation, making the system suitable for on-demand advanced toxicological testing. Humeltis’ lung tissue model is also being used to model specific diseases such as cystic fibrosis and lung cancer. This state-of-the-art technology will ultimately lead to a better understanding of pulmonary disease and drug responses, as well contribute to development of techniques to test drug responses in personalised medicine. 1

Basic Technology

The process is scaffold-free, where multiple human lung cell types placed in 3D culture condition produce their own scaffold system and form the required tissue. To obtain the necessary number of cells, once the differentiated cells have been through a sufficient period of growth, they are placed into the 3D tissue microenvironment to re-differentiate (usually within 24 hours). At this point, the primary cells begin to undergo several changes that lead to the formation of an in vitro tissue structure that closely matches that of the lung in vivo, including •• Reorganisation of the cytoskeletal architecture (Figure 1) •• A change in drug transporter gene expression, similar to that seen in vivo (Figure 2) •• Expression of cellular markers characteristic of in vivo lung tissue, e.g. surfactants (Kovacs et al., 2014) •• The down regulation of molecules that are frequently associated with de- or trans-differentiation of cell types (e.g. N-cadherin) in vitro or in given diseases (e.g. inflammatory cytokines, TNFalpha, IL1beta or IL8) compared with cells cultured in 2D (Kovacs et al., 2014) The differentiated 3D co-cultures have a cellular architecture and protein expression pattern closely matching that seen in human lung tissue in vivo, with the resulting pulmonary tissue secreting its own scaffolding components, surfactants and various other necessary proteins. The accuracy with which the model mimics the lung allows precise assessment of the complex interactions and responses triggered by test compounds, as well as enables the generation of specific disease models by using primary but diseased human lung cell types including cells from patients of cystic fibrosis, COPD, etc. Disease models are also created from a combination of healthy and disease cell lines (e.g. non-small cell lung cancer cell lines including squamous and adenocarcinoma cell lines) The drug transporter pattern is highly similar to those of the healthy primary human lung where the model contains epithelial cells, fibroblasts as well as endothelial cells.

In an attempt to construct a cell model more analogous to the in vivo microenvironment, investigators are now frequently turning to 3D cell culture. Cells grow in 3D in vivo and their particular spatial arrangement affects how they collectively respond to stimuli. In addition to benefits such as the establishment of chemical gradients more akin to an in vivo setting, 3D cell culture systems allow cells to grow in a microenvironment that enables the establishment of cell-cell interactions. How these cells interact affects their proliferation, differentiation and various other cellular functions (Bissell et al., 2003).

The Benefits of a 3D cell culture model

3D cell culture models can also be used to combine multiple cell types in a single model. Such co-cultures create an in vitro cellular environment that is more representative of the distinct and complex structures exhibited by organs and tissues. 3D co-cultures represent the next step in the development of truly accurate in vitro disease models, designed to better facilitate the translation of laboratory science into the clinic by improving the relevancy and reliability of preclinical data. Cell culture models are an integral part of the current preclinical screening process where 2D cell cultures are used in the initial screening. Although it is known that 2D cell culture suffers from a number of limitations and often fails to accurately mimic the natural environment that exists in vivo the validation of 3D cultures was performed parallel with 2D cultures to provide a clear and well characterised platform that highlighted differences in functionality, structure and drug responsiveness. Data is also comparable with results gained using primary human lung tissues. Previous studies performed by others highlight the significance and importance of such studies. For example •• A431.H9 cells grown in 3D show a higher degree of resistance to chemotherapy drugs (5-fluorouracil and tirapazamine) than cells gown in 2D under similar conditions (Tung et al., 2011) •• MCF10A cells grown in 3D exhibit higher resistance to the chemotherapy drug doxorubicin compared to cells grown in 2D (Li et al., 2010) •• MCF-7 cells grown in 3D show a higher resistance to the cytotoxic effects of the selective oestrogen receptor modulator, tamoxifen, than cells grown in 2D when exposed to the same concentration (Dhiman et al., 2005; FisherScientific, 2012) •• Cells grown in 3D spheroids better mimic the hypoxic core found in solid tumours(Tung et al., 2011), which can affect how cells respond to targeted drugs; this is important as hypoxia can enhance receptor tyrosine kinase-mediate signalling, for example, triggering the expression of genes for suppressing apoptosis or affecting DNA repair pathways (Wilson and Hay, 2011) 3

Validating Humeltis’ 3D lung tissue model

Toxicity

While the biomarker data presented so far strongly suggest that the cells growing in the Humeltis lung tissue model are behaving as they would in an in vivo environment, it is necessary to ensure that the cultured cells respond to toxic insult or other stimuli in a manner representative of the human in vivo response. This is essential for accurately modelling normal and diseased lung tissue, as well as for assessing the efficacy and safety of potential drug compounds. Toxicology studies require a reliable and accurately detectable response to stimulants. The toxicology model was validated within the non-diseased environment of the human lung model set up using healthy primary human lung cells in the presence or absence of an adenocarcinoma cell line (A549). Cell death was detected in a non-invasive manner from culture supernatants using ToxiLight™ Non-destructive Cytotoxicity BioAssay Kit (Lonza). The kit is designed to measure the release adenylate kinase (AK) enzyme from damaged cells. AK is a robust protein present in all eukaryotic cells, which is released into the culture medium when Cellular responses were tested against individual and combinations of toxic and non-toxic compounds using three time points (0, 24, 72 hrs), at five different concentrations. Results using the test system clearly indicate that 2D mono-cultures are more sensitive to cisplatin toxicity than 2D co-cultures – even more so than 3D co-cultures (Figure 3). Non-toxic agents did not affect cellular viability in either 2D or 3D cell culture conditions (Figure 3), while combination of cisplatin and Ko143 for example (an inhibitor of ABCG2 drug transporter) induced a well-defined increase in cellular toxicity to cisplatin in 3D culture conditions.

Disease Models

The efficacy of drugs cannot rely on toxicology studies alone. It is therefore necessary to establish models of specific disease environments.

Induced inflammation To assess the capability of the Humeltis lung tissue model to reproduce an inflammatory response, it was exposed to both a known endotoxin, lipopolysaccharide (LPS), which is released from the cell wall of all gram-negative bacteria) and cigarette smoke. Endotoxins are known pro-inflammatory molecules (Bannerman and Goldblum, 1999), while a wide range of studies have demonstrated the ability of cigarette smoke to induce inflammation and oxidative stress (van der Vaart et al., 2004).

Results from the Humeltis lung tissue model showed that cell cultures exposed to 1 µg/L LPS over 72hr (Figure 4A) or to repeated cigarette smoke exposure (15 minutes/day over five days in a standard smoking chamber using commercially available standardised cigarettes) exhibited signs of an inflammatory response (Figure 4B). This was demonstrated by an up-regulation of molecules such as Wnt5a, which has been shown to stimulate inflammation (Kovacs et al., 2014) and inflammatory cytokine expression. These datademonstrated that both LPS and cigarette smoke triggered an inflammatory response in the model, as happens in vivo.

Immune response Recent research suggests that the response to a particular drug is variable with respect to the state of the host immune system, e.g. in the case of chemotherapeutics, being immunocompetent versus immunodeficient (Bracci et al., 2014). In an in vitro setting, it has also been shown that co-culturing tumour cells with macrophages will modify how a model responds to certain chemotherapeutics (Bracci et al., 2014; Bryniarski et al., 2009; Buhtoiarov et al., 2011; Potapov et al., 1988). It is therefore important that in vitro models include the addition of functionally active immune cells in order to observe the types of drug response representative of those seen in vivo. For this reason, the Humeltis lung models can include macrophages, neutrophils and T-cells to better reflect the native cell composition of the lung and thus provide a more accurate assessment of the likely drug response seen during clinical trials. Macrophage-containing tissue systems could be particularly important in inflammation as well as in cancer drug responses, as macrophages are integral part of the tissue microenvironment of the lung and immune modulated environments. The novel concept of macrophage differentiation into a classically activated phenotype (M1) and an alternatively activated phenotype (M2), the role of tumour-associated macrophages (TAMs) makes the presence of macrophages in tissue models even more essential. In human malignant tumours, type M2 macrophages (TAMs) act as tumour-promoting macrophages and contribute to angiogenesis, immunosuppression and activation of tumour cells. There are two sources of monocytes/macrophages in the model: •• U937, a pro-monocytic cell line that can be differentiated towards monocytes •• Monocytes purified from human peripheral blood mononucleated cells (PBMC) There are also two sources of neutrophils: •• HL60, a myelomonocytic cell line that can be differentiated towards neutrophils •• Primary human neutrophils purified from PBMC Primary human monocytes that were differentiated in vitro to macrophages and were pre-activated by 1 mg/ml LPS, than cultured in the model were able to destroy the lacy structure of the lung tissue within 72 hrs.

Cancer Modelling lung cancer can aid development of more effective lung cancer drugs. Several types of lung cancer models have been developed by Humeltis. •• Using cancer cell lines | Various lung cancer cell lines (adenocarcinoma,

squamous cell carcinoma, etc.) can be included in the model as diffuse or solid tumour-like tissues amongst normal cells â&#x20AC;˘â&#x20AC;˘ Using primary cancer cells | Primary lung cancer cells can be cultured in a 3D environment with non-cancerous lung cell types Using cancer cell lines in the model can provide a robust, reliable and reproducible test system as a first screen for anticancer drugs. Humeltis created an adenocarcinoma cell line expressing green fluorescent protein (A549-GFP) for flow cytometric analysis-based screening of cancer drug efficacy. In such systems, the carcinoma cells are set amongst normal lung cancer cells in 2D and 3D co-culture conditions (Figure 5).

Modulation of gene expression

In order to accurately model certain physiological processes, disease states and drug responses, it can be useful to modify gene expression or to interfere with a particular signalling pathway (for example to trigger a desired phenotype or even alleviate the effects of a disease). When using the Humeltis lung tissue model, gene expression levels can be modulated using recombinant viruses, siRNAs or via pharmacological activation/inhibition, before or after the tissue structure has been created. As an example, gene expression has been altered using recombinant adenoviruses (rAd). rAds included a control and one carrying beta-catenin-interacting protein, ICAT, which negatively regulates gene expression. The successful inhibition of the beta-catenin-dependent pathway was illustrated by the up-regulation of pro-surfactant protein C in our recently published article (Kovacs et al., 2014), showing how Wntsignalling regulates molecular events that lead to pulmonary senescence. In particular, Wnt4 and Wtn5a levels were shown to be increased in the ageing lung leading to an increase in myofibroblast-like differentiation, which could be linked to the active control and mitigation of tissue damage (Kovacs et al., 2014).

Efficacy

Drug efficacy studies are important to show specific effects of drugs on individual cell types within a complex tissue environment. To be able to monitor the effects of anticancer drugs in lung cancer for example, a flow-cytometric assay was developed by Humeltis, where total cell numbers of A549-GFP and unstained non-cancerous cells can be monitored separately. The two main population of cells (GFP+ and GFP-) can also be analysed by a 7AAD based apoptosis assay. Cell viability measured by 7-amino-actinomycin D (7AAD) staining is shown in Figure 6. The assay developed by Humeltis provides additional information to the ToxiLight toxicity assay. 7-AAD stains non-viable cells in flow cytometric analysis and can be used in combination with PE (phycoerythrin) and FITC (fluorescein isothiocyanate)-conjugated antibodies or GFP (green fluorescent protein) in 2-colour analysis, therefore cell death can be specifically investigated in subpopulations of cells within the tissue.

The lung tissue model has been successfully validated as an accurate in vitro model of pulmonary tissues that closely mimics how lung tissues respond in vivo to various stimuli, making it a powerful tool for an array of toxicity and efficacy studies.

Humeltis’ 3D lung model in practice

Based on the model, the Lungetox™ assay can be used to quickly (0–72 hours) test for •• •• •• ••

Macrophage activation Cellular toxicity Mitochondrial impairment Apoptosis and carcinogenicity

While the Lungeffix™ assay is an effective way of investigating drug efficacy. Researchers at Humeltis have also developed a flow-cytometric assay that provides a reliable workflow for comparing the cytostatic and cytotoxic effects of chemotherapeutic agents. In addition to modelling the effects of drug candidate molecules, the Humeltis lung system can also provide a functional representation of several pulmonary diseases. A detailed understanding of disease pathology and progression at the molecular level is required in order to construct a model that will yield useful results. Humeltis’ 3D lung cell culture model is capable of modelling •• •• •• •• •• ••

Inflammation Inducing inflammatory processes Using primary human cells of pulmonary inflammation (e.g. COPD) Cystic fibrosis (CF) Lung cancers Various lung cancer cell lines included as diffuse or solid tumour-like tissues amongst normal cells •• Primary lung cancer cells cultured in a 3D environment with non-cancerous lung cell types

Humeltis’ 3D lung model not only constitutes a flexible model for assessing the effects of novel candidate drugs, but also allows for an accurate reconstruction of complex lung disease states in vitro. Models such as this greatly enhance our understanding of disease pathology and make it easier to develop effective treatments that are more likely to perform as expected during clinical trials. While animal models have been useful in the past, they cannot provide enough information at the cellular level. As an example, mouse disease models have been shown to suffer from poor transferability to the human disease state, as they are unable to account for the numerous genetic polymorphisms present within a human population (Seok et al., 2013).

A welcomed improvement to preclinical drug testing

To move our understanding forward, Humeltis are compelled to utilise models such as the lung tissue model described here in order to better elucidate how diseases develop and how drugs might act to provide effective treatment. Improving our understanding during preclinical stages can go a long way to reducing the chances of late stage drug trial failure, helping to reduce costs, save time and increase efficiency during drug development. Ultimately, our shared aim is to improve the lives of patients by developing therapeutics that treat disease more effectively with less unexpected side effects. We believe that models such as the Humeltis 3D lung cell culture model are an important tool for achieving these aims and can support the pharmaceutical industry in its mission to alleviate the economic and personal burdens imposed by lung disease. For more information about the benefits, applications and technical details of the Humeltis lung tissue models, please visit our website or contact us:

Address: Tel.: Web: E-mail:Â

H-7614 PĂŠcs 14. Pf. 66. +36 30 915 7630 www.humeltis.com info@humeltis.com

Bannerman, D. D. and Goldblum, S. E. (1999). Direct effects of endotoxin on the endothelium: barrier function and injury. Lab. Invest. 79, 1181–1199.

References

Bissell, M. J., Rizki, A. and Mian, I. S. (2003). Tissue architecture: the ultimate regulator of breast epithelial function. Curr. Opin. Cell Biol. 15, 753–762. Bracci, L., Schiavoni, G., Sistigu, a and Belardelli, F. (2014). Immune-based mechanisms of cytotoxic chemotherapy: implications for the design of novel and rationale-based combined treatments against cancer. Cell Death Differ. 21, 15–25. Bryniarski, K., Szczepanik, M., Ptak, M., Zemelka, M. and Ptak, W. (2009). Influence of cyclophosphamide and its metabolic products on the activity of peritoneal macrophages in mice. Pharmacol. Reports 61, 550–557. Buhtoiarov, I. N., Sondel, P. M., Wigginton, J. M., Buhtoiarova, T. N., Yanke, E. M., Mahvi, D. A. and Rakhmilevich, A. L. (2011). Anti-tumour synergy of cytotoxic chemotherapy and anti-CD40 plus CpG-ODN immunotherapy through repolarization of tumour-associated macrophages. Immunology 132, 226–239. Dhiman, H. K., Ray, A. R. and Panda, A. K. (2005). Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen. Biomaterials 26, 979–986. FisherScientific (2012). Routine Assessment of Cancer Cell Cytotoxicity in a Novel Three Dimensional Culture Assay. Kovacs, T., Csongei, V., Feller, D., Ernszt, D., Smuk, G., Sarosi, V., Jakab, L., Kvell, K., Bartis, D. and Pongracz, J. E. (2014). Alteration in the Wnt microenvironment directly regulates molecular events leading to pulmonary senescence. Aging Cell 13(5): 838–849.

Li, Q., Chow, A. B. and Mattingly, R. R. (2010). Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors. J. Pharmacol. Exp. Ther. 332, 821–828. Potapov, S. L., Korman, D. B., Shamaev, V. I., Ershova, R. B. and Makarov, O. V (1988). Sensitivity of clonogenic cells of human ovarian ascitic cancer to antitumor drugs. Arch. Geschwulstforsch. 58, 99–104. Seok, J., Warren, H. S., Cuenca, A. G., Mindrinos, M. N., Baker, H. V, Xu, W., Richards, D. R., McDonald-Smith, G. P., Gao, H., Hennessy, L., et al. (2013). Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc. Natl. Acad. Sci. U. S. A. 110, 3507–12. Tung, Y.-C., Hsiao, A. Y., Allen, S. G., Torisawa, Y., Ho, M. and Takayama, S. (2011). High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst 136, 473–478. Van der Vaart, H., Postma, D. S., Timens, W. and ten Hacken, N. H. (2004). Acute effects of cigarette smoke on inflammation and oxidative stress: a review. Thorax 59, 713–721. Wilson, W. R. and Hay, M. P. (2011). Targeting hypoxia in cancer therapy. Nat. Rev. Cancer 11, 393–410.

Figures

Figure 1: The actin cytoskeleton of cells in 2D and 3D. Humeltisâ&#x20AC;&#x2122; lung tissue model (5) mirrors the cytoskeleton of cells in the human lung in vivo (4). 1: SAEC (small airway epithelial cells); 2: HMVEC-L (human vascular endothelial cell lung); 3: NHLF (normal human lung fibroblast).

Figure 2: Drug transporter mRNA expression patterns in 2D mono-cultures and 3D co-cultures of various cellular composition. The Humeltis lung tissue model mirrors the selected drug transporter expression pattern of cells in the human lung in vivo. SAEC: small airway epithelial cells, HMVEC-L: human vascular endothelial cell lung, NHLF: normal human lung fibroblast.

Figure 3: Cell viability studies using ToxiLight assay. Cultures were set up as triplicates and treated with 9 µg/ml cisplatin and/or with Ko143 ABCG2 inhibitor at the concentration of IC50 (25 µM) for 72 hrs. Adenylate kinase activity levels were measured from cell culture supernatants using a bioluminescent firefly luciferase reaction and calculated against a standard curve, then plotted as % of viability.

Figure 4: The lung model displays an inflammatory response when exposed to lipopolysaccharide (LPS). A) Control tissue, B) LPS exposed tissue. Wnt5a (green) is an activator of NFκB and therefore gene expression associated with NFκB activity. KRT7 (red) is an epithelial cell marker. The nuclei were stained blue with DAPI. (A, B). Inflammatory cytokine mRNA levels increase upon cigarette smoke exposure of the model. mRNA levels were compared to un-treated control tissues (C).

Figure 5: Cells labelled with green fluorescent protein (GFP) can be visualised easily by microscopy methods or flow cytometry. 1: 2D co-culture of fibroblasts (NHLF) and cells of the A549 adenocarcinoma cell line. 2: 2D co-culture of fibroblasts (NHLF) and cells of the A549-GFP (green) adenocarcinoma cell line. 3: 3D co-culture of fibroblasts (NHLF) and A549 cells. 4: Confocal microscopy picture of a 3D co-culture. Fibroblasts (NHLF) are red (pre-stained with a fluorescent physiological dye) and A549-GFP cells are green.

Figure 6: The effect of cisplatin on GFP+ A549 adenocarcinoma cells amongst non-cancerous, primary human fibroblasts (NHLF) in 2D and 3D co-cultures

......................................................................................

Notes

...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ...................................................................................... ......................................................................................

......................................................................................

Address: Tel.: Web: E-mail:Â

H-7614 PĂŠcs 14. Pf. 66. +36 30 915 7630 www.humeltis.com info@humeltis.com