Modeling of lung organoid-based fibrosis for testing the sensitivity of anti-fibrotic drugs

Abstract

Background

Pulmonary fibrosis models play crucial roles in research of pulmonary fibrosis and anti-fibrosis drug screening. Despite the establishment of several pulmonary fibrosis models including lung fibrosis animals, stem cell differentiation, pulmospheres and so on, the one that mimic the personalized native lung lacks.

Methods

We here developed a lung organoid-based fibrosis (LOF) model from native lung tissue, and the potential of the LOFs for the sensitivity test of anti-fibrotic drugs was evaluated.

Results

Our results showed that the LOFs could be self-organized from the lung organoids and the fibroblasts derived from native lung tissue. Histochemical examination demonstrated that the LOFs were characteristic of pulmonary fibrosis in structure. Single-cell sequencing (SCS) further revealed that the cell clusters mimicked fibrotic process at cellular and molecular levels in the LOFs. Drug sensitivity test indicated that the LOFs could not only be used to evaluate the efficacy of anti-fibrotic drugs, but also display their toxicity.

Conclusions

We demonstrate that the LOFs represent an efficient fibrotic model that mimics faithfully the personalized characteristics of native lung tissue.

Introduction

Pulmonary fibrosis is an interstitial lung disease characterized by progressive scarring of lung tissue which reduces gas exchange, and leads to progressive respiratory failure [1]. Investigation into the mechanism and drug screening depends strongly on fibrotic models that mimic the structure and development of pulmonary fibrosis [2,3,4]. However, pulmonary fibrosis is a very complex process, and not only are the pathogenic factors various, but also the cells in which participate are multiple [4]. Genetic inheritance, environment, virus and so on all can initiate pulmonary fibrosis [5, 6]. Several cell types including lung epithelial cells, fibroblasts, airway basal cells are implicated in the development and they interact and aberrant alteration of multiple singling molecules and pathways is involved in the development of pulmonary fibrosis [1, 7]. Pulmonary fibrosis has its own unique personal cellular and molecular characteristics, and therefore modeling the pathogenetic processes has been more challenging.

Several pulmonary fibrosis models including lung fibrosis animals, fibrotic lung organoid based on stem cell differentiation, pulmospheres and so on have been developed [2, 8,9,10]. Lung fibrosis animals induced by bleomycin display main features of pulmonary fibrosis, and have been widely used for research of pulmonary fibrosis and anti-fibrosis drug screening [2]. Differentiated lung fibrosis can be obtained by manipulating genes related to pulmonary fibrosis in pluripotent stem cell differential models, and they can be used to model specific fibrotic lung disease [8, 9]. Pulmospheres are 3-dimensioanl spheroids, which can be self-assembled from the cells derived from native lung, and the fibrotic pulmospheres can be induced by TGF-β [10]. Due to the lung origin, pulmospheres carry personalized information of pulmonary cells, but lack 3-dimensional structural characteristics of pulmonary fibrosis.

Lung organoids that self-assemble from dissociated lung cells of native lung tissue recapitulate histological architecture, gene expression and genomic landscape, preserving the characteristics of original organ [11, 12]. Multiple lung epithelial cells including club cells, Goblet cells, ciliated cells and so on have been demonstrated to participate in the self-assemble of lung organoids, and they arrange properly reflective of those in situ in lungs [13]. These results indicate that lung organoids from native lung tissue are characteristics of their native structural features. we therefore hypothesized that re-organization of pulmonary fibrosis derived from lung organoids and fibroblasts can mimic the characteristics of original tissue. In the present study, we set up a lung organoid-based fibrosis model by co-culturing lung organoids with lung-derived fibroblasts, and the potential of lung organoid-based fibrosis model for anti-fibrosis drug sensitivity was evaluated.

Materials and methods

Animals

Male C57BL/6 mice (> 8 weeks old) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd (Beijing, China). A total of five mice were used in our study. All animal experiments involved in this study were approved by the Ethics Committee of Darkjade Sciences,Inc.

Culture of lung organoids and fibroblasts

Isolation of primary lung organoid cells from the lungs after euthanasia of C57BL/6 mice with carbon dioxide. Fresh mouse lung tissue was washed three times using PBS containing 1xpenicillin/streptomycin, and then was minced using scissors in a 6 cm dish with digestion buffer (0.1% Collagenase Type IV in DMEM/F12 supplemented with 10% fetal bovine serum). After half an hour’s digestion, the tissue was pipetted up and down several times to release cells. The mixture was passed through a strainer to remove the debris. The cells were then plated onto 6-well culture plates for 10–20 min to allow the attachment of the fibroblasts. Then, the suspension was collected and re-suspended in organoid culture medium. After mixed thoroughly with Matrigel, the cells/Matrigel was plated onto 24-well ultra-low adherence plates for lung organoid culture. The organoid medium was DMEM/F12 supplemented with 100 ng/ml of FGF10 (OrganRegen, Shandong, China), 50 ng/ml human EGF (OrganRegen, Shandong, China), B27 (Invitrogen, MA USA), 1μMA8301(MCE, Shanghai, China), 10 μM ROCK inhibitor (MCE, Shanghai, China), and 1% penicillin/streptomycin (BasalMedia, Shanghai, China). For the expansion of fibroblasts, the cells that attached on the plates were expanded in medium (DMEM supplemented with 10% fetal bovine serum). The medium was changed every other day, and the cells were passaged when they reached 80% confluence.

Self-organization of the LOFs

The cells from the P3–P5 lung organoids were re-suspended in organoid culture medium for the self-assemble of the LOFs. Depend on the co-culture phase of the fibroblasts and the lung organoids, two approaches of the O–F and the C–F were set up. In the O–F model, the lung organoids were formed before coculture, and then the fibroblasts were added to coculture with the lung organoids. The cells from lung organoids were mixed together with the fibroblasts to form the LOFs (C–F model). The ratio of the cells from the lung organoids and the fibroblasts was 1:3.

Scanning electron microscope observation

Samples were fixed in by electron microscopy fixative for 2 h at room temperature, and post-fixed for another 1 h at room temperature in 1% OsO4 at room temperature. After wash in 0.1M PB (pH 7.4) for 3 times, the samples were dehydrated with a graded series of ethanol by a routine method and critical point dried with CO₂ using a critical point dryer. The samples were attached to metallic stubs using carbon stickers and sputter-coated with gold for 30 s, and observed and took images with scanning electron microscope.

Morphological examination

The lung organoids or the LOFs were fixed with 10% neutral formalin overnight, paraffin embedded and sectioned (4–5 m). Sections were deparaffinized and stained with H&E for histological analysis. For immunohistochemical staining, the sections were carried out using the following primary antibodies: anti-CK7 (1:1000), anti-a-SMA (1:1000), anti-vimentin (1:1000), anti-desmin (1:1000). All secondary antibodies were used at 1:500. The images were captured using MShot Image Analysis System.

Single cell sequencing

The lung tissue, the lung organoids or the LOFs were subjected to enzyme digestion and dissociated into single cell suspension using digestive buffer. Individual cells are isolated using a single cartridge together with UMI-barcoded magnetic mRNA capture-beads based on the BD Rhapsody platform. The mRNAs are captured and retrieved together with the beads out of the microwells upon cell lysis, and pooled into a single tube. The RT step is performed within a single tube, and whole transcriptome amplification (WTA) and the sequencing libraries were generated according to the BD Rhapsody single-cell whole-transcriptome amplification workflow.

Drug testing

The LOFs from the O–F or the C–F were treated by the drugs of Pirfenidome and Nintedanib for three days. For each drug, the concentration was set in triplicate. The images of the LOFs were captured every day under phase-contrast microscope. At the end of the treatment, the LOFs were subjected to histochemical examination for H&E and immunohistochemical staining.

Statistical analysis

Statistical analysis was performed with the GraphPad Prism 7 software. Data presented were from at least three independent experiments. The data were expressed as mean ± standard deviation (SD). Student’s t-test or ANOVA tests were used to compare the difference among groups, *P < 0.05 indicated a significant difference.

Results

Self-assemble of the LOFs

The LOFs self-assembled from the lung organoids and the fibroblasts derived from native lung tissue. To obtain the lung organoids and the fibroblasts, fresh lung tissue was dissociated into single cells and cultured for the lung organoids or the fibroblasts, respectively. The lung organoids were formed after two days’ culture, and their sizes increased gradually (Fig. 1A). Passage could be continually performed every 3–4 days (Fig. 1A). Immunohistochemical staining demonstrated that the lung organoids were positive for CK7, but negative for α-SMA, desmin and vimentin (Fig. 1A). The expanded fibroblasts displayed spindle-like shapes, and were positive for vimentin, and partially for a-SMA (Fig. 1B). The assemble of the LOFs was achieved by coculturing the lung organoids with the fibroblasts. The lung organoids or organoid-derived cells self-assembled into the LOFs on the second day after combination with the fibroblasts in either the C–F or the O–F (Fig. 1C), and the shape of the formed LOFs varied with the methods that were used. In the O–F, the lung organoids congregated each other to form the LOFs, in which their shapes were maintained, and the fibroblasts distributed in the interstitial of the lung organoids (Fig. 1C). In contrast, the organoid-derived cells reorganized with the fibroblasts and formed single blunt LOFs whose shapes of lung organoids were not visible any more in the C–F model (Fig. 1C). The sizes of the LOFs increased gradually over time in both the C–F and the O–F model. These results indicated that the LOFs could be fabricated from the native lung tissue by assembling.

Fig. 1

Self-assemble of the LOFs. A Microscope photography of the lung organoids and their morphological characteristics. Immunohistochemical staining of lung organoids for CK7, vimentin, desmin and a-SMA, showing that the lung organoids consisted of the CK7-positive cells. B Microscope photography of the fibroblasts and their morphological characteristics. Immunofluorescent staining for vimentin and a-SMA. C Microscope photography of the LOFs, showing the different shapes of the LOFs that formed in the C–F and O–F model

Morphological characteristics of the LOFs

To delineate the morphological characteristics of the LOFs, scanning electron microscope (SEM) observation was performed. Compared to the tightly packaged lung organoids, the LOFs in both the O–F and the C–F was larger and more irregular (Fig. 2A). Additionally, the surface of the LOFs was separated into small parts in the C–F (Fig. 2B). Histological examination further revealed that the LOFs were 3-dimensioanl cell masses that mainly consisted of the lung organoids surrounded by the interstitial cells (Fig. 2B). The lung organoids maintained their multiple chamber-like morphology and formed island-like tissue, and the interstitial cells distributed between the tissue in the O–F (Fig. 2B). In contrast, larger, reticulate LOFs surrounded by interstitial cells, which consisted of epithelial cells, were formed when the lung organoid-derived cells mixed with the fibroblasts to coculture in the C–F (Fig. 2B). Immunohistochemical examination showed that the LOFs were consisted of CK7-positive organoids surrounded by the vimentin-positive fibroblasts (Fig. 2C). The fibroblasts could also be found in the inner area of the LOFs in the C–F. The distribution of the fibroblasts was different between the O–F models and the C–F models (Fig. 2C). The fibroblasts were dispersed in the interstitial of the lung organoids in the O–F (Fig. 2C). In contrast, the fibroblasts were in the outer layer of the LOFs in the C–F models (Fig. 2C). These results demonstrated that the LOFs were characteristic of lung fibrosis in histology.

Fig. 2

Morphological characteristics of the LOFs. A, Scanning electron microscope observation of the lung organoids and the LOFs cultured in the O–F or C–F, showing the different shapes of the LOFs. B, H.E. staining of the LOFs cultured in the O–F or C–F, showing that the formation of the island-like (O–F) or reticulate (C–F) tissue surrounded by the interstitial cells. C, Immunohistochemical staining of the LOFs cultured in the O–F or C–F for CK7, vimentin and a-SMA, showing that the CK7-positive cells surrounded by the vimentin-positive cells and the activated a-SMA positive fibroblasts

Single-cell sequencing of the LOFs

The cell components of the LOFs were analyzed using single-cell sequencing. The LOFs contained 7 cell components including mature type l alveolar pneumocytes (g-0 cluster), damage-associated transient progenitors (g-2 cluster), fibroblasts (g-3 cluster) and so on, which were involved in the cell components of the native lung tissue (Fig. 3A). Notably, the ratios of the fibroblasts (g-3 cluster) and the damage-associated transient progenitors (g-2 cluster) to mature type l alveolar pneumocytes (g-0 cluster) were higher in the LOFs than the one in the native lung (Fig. 3B). Go analysis for the biological process, cellular components and molecular function sections further showed that the keratinization was enhanced in the LOFs compared to the one in the native lung (Fig. 3C). Accordingly, several metabolic processes such as glutathione metabolism were diminished in the LOFs (Fig. 3C). Additionally, the mesenchymal cell differentiation and development as well as the synthesis of extracellular matrix and organization was enhanced in the LOFs compared to the organoids (Fig. 3C). These results indicated specifically the characteristics of lung fibrosis in cell components.

Fig. 3

Single-cell sequencing of the mouse lung, lung organoid and the LOFs. A, UMAP plot of cell clusters, showing that the LOFs including key cell clusters of lung fibrosis. B, Histograms of cell clusters, showing that the LOFs had higher ratios of the fibroblasts (g-3 cluster) and the damage-associated transient progenitors (g-2 cluster) to mature type l alveolar pneumocytes (g-0 cluster) compared to the native lung. C, Column charts of Go analysis for the biological process, cellular components and molecular function sections, showing that the keratinization was enhanced in the LOFs compared to the one in the native lung

Cell communication of cell clusters was further analyzed in the LOFs, and strong interactions existed between the proliferative mesenchymal progenitors (g-1) and fibroblasts (g-3) and other cell clusters (g-0, g-2 and g-5) (Fig. 4A). The signaling molecules from the proliferative mesenchymal progenitors (g-1) and fibroblasts (g-3) mainly consisted of FGF, ncWNT and so on, and the receivers were mature type l alveolar pneumocytes (g-0), damage-associated transient progenitor (g-2) and themselves (Fig. 4B). Among the signaling pathways, the ncWNT and TGF-β were the most important ones in the network. The signaling molecules of ncWNT, which were mainly from the proliferative mesenchymal progenitors (g-1) and fibroblasts (g-3) produced effects on the mature type l alveolar pneumocytes (g-0), damage-associated transient progenitor (g-2) and themselves (Fig. 4C). The signaling molecules from the proliferative mesenchymal progenitors (g-1) in the TGF-β pathway acted on the mature type l alveolar pneumocytes (g-0) and itself (Fig. 4C, D). These results revealed the potential interaction in the fibrotic lung.

Fig. 4

Cell communication of cell clusters in the LOFs. A, Neuron network graph of interactions, showing the potential interactions of different cell clusters. B, Heatmap of signaling pathway network, showing the outgoing singling patterns and incoming signaling patterns. C, Heatmap of signaling pathway network, showing the interactions of ncWNT and TGF-β signaling pathway network in the LOFs. D, Bubble plot of TGF-beta ligand–receptor interactions between different cell clusters

Drug sensitivity test using the LOFs

We further evaluated the potential of the LOFs for testing the sensitivity of anti-fibrosis drugs. Two anti-fibrosis drugs of Pirfenidome and Nintedanib that were used in the clinic were examined using the LOFs, and the morphological alteration of the organoids and the fibroblasts in the LOFs was evaluated. Both Pirfenidome and Nintedanib could inhibit the growth of the fibroblasts as evidenced by the reduction of the interstitial cells between organoids, and the effects were concentration- and time-dependent (Fig. 5A, B). There exist differences in the change of the LOF shapes in response to drug treatment between the C–F and O–F. The outline of the LOFs gradually restored to the shape of the lung organoids after treatment with drugs due to the reduction of the fibroblasts in the O–F (Fig. 5A, B). In contrast, the blunt outline of the LOFs displayed the lung organoid-like shape in the C–F (Fig. 5A, B). Immunohistochemical staining demonstrated that the vimentin-positive fibroblasts decreased over the increase of drug concentration (Fig. 5C, D). Notably, the effects of the drugs on the growth of lung epithelial cells could also be examined in the LOFs. The sizes of the organoids that mainly consisted of epithelial cells in the LOFs treated by Nintedanib weren’t influenced even at the highest concentration of 20uM (Fig. 5C, D). In contrast, the sizes of the organoids in the LOFs treated by Pirfenidome were severely reduced over the increase of drug concentration (Fig. 5A, B). These results indicated that the LOFs could display the efficacy and toxicity of the anti-fibrosis drugs.

Fig. 5

Sensitive evaluation of the anti-fibrosis drugs of Pirfenidome and Nintedanib using the LOFs. A, Light microscope of the LOFs treated by Pirfenidome in the C–F and O–F, showing that the number of the fibroblasts decreased over the increase of Pirfenidome concentration. B, Light microscope of the LOFs treated by Nintedanib in the C–F and O–F, indicating that the inhibitory effects of both drugs on the fibroblasts could be observed. C, Morphological characteristics of the LOFs treated by Pirfenidome with HE staining and immunohistochemical staining for CK7 and vimentin in the C–F. D, Morphological characteristics and quantitative analysis of the LOFs treated by Nintedanib with HE staining and immunohistochemical staining for CK7 and vimentin in the C–F. showing that Pirfenidome influenced the growth but Nintedanib didn’t. Data are representative of more than three independent experiments. All values are mean ± s.d. Significance was calculated by one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test; ***P < 0.001

Discussion

Pulmonary fibrosis has its own unique personalized characteristics, and this necessitates the development of individual pulmonary fibrosis model [14]. Self-assemble from native lung uses the individual cells to generate 3-dimensional pulmonary fibrosis model that is characteristic of personalized features. Pulmosphere culture forms 3-dimensional tissue mass composed of cells derived exclusively from primary lung biopsies and inclusive of lung cell types reflective of those in situ by driving cells from native tissue to aggregate each other by culture on the ultra-adherence surface [10]. In the present study, we developed a LOF from native lung tissue based on pulmonary organoids and their corresponding fibroblasts of native lungs. Due to the organoid recapitulation of histological architecture, gene expression and genomic landscape and re-organization of both the organoids and the fibroblasts, the LOFs represent lung fibrosis models that can mimic the structural and functional characteristics.

Key features of pulmonary fibrosis are the activation and proliferation of fibroblasts resulting in the deposit of ECM and subsequently wrap up the alveolus pulmonis in the interstitial of pulmonary alveoli [4]. In either the fibrotic lung organoid based on stem cell differentiation or the 3D pulmosphere models, the fibroblasts exist in the 3-dimensioanl tissue mass but the lung fibrosis-like structural morphology is not formed [8,9,10]. In contrast, the LOFs mimic more closely the lung fibrosis in structure as evidenced by the following findings: 1) The LOFs form pulmonary fibrosis-like structure consisting of the lung organoids surrounded by the fibroblasts. 2) Both the vimentin-positive and the α-SMA-positive cells are found in the LOFs. 3) The LOFs mimics fibrotic process as evidenced by the higher ratios of the fibroblasts (g-3 cluster) and the damage-associated transient progenitors (g-2 cluster) to mature type l alveolar pneumocytes (g-0 cluster), the keratinization of their mature type l alveolar pneumocytes (g-0 cluster) as well as the interactions between the cell clusters. It’s noteworthy that the structure of the LOFs varies with the used coculture approaches. The sizes and shapes of the lung organoids in the LOFs are maintained in the O-F models, and the fibroblasts distribute loosely around the organoids, while the lung organoid-derived cells reorganize and form larger, reticulate organoids tightly surrounded by the fibroblast layers.

The LOFs can potentially be used to test the drug sensitivity or screen anti-fibrosis drugs. The inhibitory effects of anti-fibrosis drugs can be evaluated by inhibiting the invasion of the fibroblasts induced by TGF-β in the pulmospheres [10]. In our LOFs, the anti-fibrosis effects of the tested drugs can be directly observed by the reduction of the fibroblasts in the LOFs and is further demonstrated by histochemical alterations. Moreover, the toxicity of drugs to epithelial cells can also be efficiently evaluated by the LOFs, and the growth of the LOFs is inhibited by the drugs that are poisonous to the cells as demonstrated by the growth of organoids. For the test of Pirfenidome and Nintedanib, our results indicate that Pirfenidome influences the growth of epithelial cells but Nintedanib doesn’t though both of them can inhibit the growth of fibroblasts, indicating that excessive use of Pirfenidome for the treatment of pulmonary fibrosis may do harm to lungs. The LOFs may also be used as models for research of pulmonary fibrosis. It’s noteworthy that the methods can be used for the self-assemble of human LOFs although all the experiments are performed based on the cells derived from mouse lung tissue.

Conclusions

We set up a novel lung fibrosis model of LOF based on lung organoids and fibroblasts. Our results demonstrate that the LOFs self-assembling from the organoids and the fibroblasts derived from native lung mimic the characteristics of lung fibrosis and represent personalized models. The LOFs can be used as models for drug screening of anti-fibrosis drug and sensitivity test of individual drugs.