Use of a pulmosphere model to evaluate drug antifibrotic responses in interstitial lung diseases

Abstract

Background: Interstitial lung diseases (ILD) encompass a heterogenous group of diffuse parenchymal lung disorders characterized by variable degrees of inflammation and fibrosis. Pretherapeutic clinical testing models for such diseases can serve as a platform to test and develop effective therapeutic strategies. In this study, we developed patient derived 3D organoid model to recapitulate the disease process of ILDs. We characterized the inherent property of invasiveness in this model and tested for antifibrotic responses with an aim to develop a potential platform for personalized medicine in ILDs.

Methods: In this prospective study, 23 patients with ILD were recruited and underwent lung biopsy. 3D organoid-based models (pulmospheres) were developed from the lung biopsy tissues. Pulmonary functioning testing and other relevant clinical parameters were collected at the time of enrollment and follow up visits. The patient derived pulmospheres were compared to normal control pulmospheres obtained from 9 explant lung donor samples. These pulmospheres were characterized by their invasive capabilities and responsiveness to the antifibrotic drugs, pirfenidone and nintedanib.

Results: Invasiveness of the pulmospheres was measured by the zone of invasiveness percentage (ZOI%). The ILD pulmospheres (n = 23) had a higher ZOI% as compared to control pulmospheres (n = 9) (516.2 ± 115.6 versus 54.63 ± 19.6 respectively. ILD pulmospheres were responsive to pirfenidone in 12 of the 23 patients (52%) and responsive to nintedanib in all 23 patients (100%). Pirfenidone was noted to be selectively responsive in patients with connective tissue disease related ILD (CTD-ILD) at low doses. There was no correlation between the basal pulmosphere invasiveness, response to antifibrotics, and FVC change (Δ FVC).

Conclusions: The 3D pulmosphere model demonstrates invasiveness which is unique to each individual subject and is greater in ILD pulmospheres as compared to controls. This property can be utilized to test responses to drugs such as antifibrotics. The 3D pulmosphere model could serve as a platform for the development of personalized approaches to therapeutics and drug development in ILDs and potentially other chronic lung diseases.

Fig. 1

ILD lung pulmospheres are characterized by an invasive phenotype compared to controls. A Representative image of an invasive pulmosphere (ILD- top panel, control -bottom panel) depicting total area of invasion (purple) and inner core area (yellow). B Formula quantitating ZOI. C Scatter plot of control pulmospheres vs ILD pulmospheres depicting significantly higher invasiveness of the ILD pulmospheres n = 9 for control and 23 for ILD (Control Vs ILD, 54.63 ± 19.6 vs 516.2 ± 115.6, ***P value < 0.0001 estimated by Mann–Whitney test). D Simple linear regression analysis of ZOI% (invasiveness) with Δ %FVC (change in %FVC) showing positive correlation, P value < 0.001, R² = 0.64, r = 0.80

Fig. 2

Fibronectin expression in increased in ILDs and correlates with pulmosphere invasiveness. A IHC staining showing expression of fibronectin in formalin-fixed, paraffin-embedded tissue section of a representative of 9 control subjects and 5 subjects with ILD. Scale bars: 100 μm. B Scatter plot of DAB fibronectin-EDA intensity control vs ILD tissue depicting significantly higher intensity in the ILD tissues. N = 9 for control subjects and 17 for ILD (Control Vs ILD, 0.075 ± 0.020 vs 0.122 ± 0.033, **P value = 0.0012 estimated by Mann–Whitney test). C Simple linear regression analysis of ZOI% (invasiveness) with fibronectin expression showing positive correlation, P value < 0.001, R² = 0.88, r = 0.94. D RT-PCR showing col1A1 mRNA expression normalized to β-actin among Control Vs ILD pulmospheres,0.73 ± 0.06 vs 0.97 ± 0.31, P value = 0.0476 estimated by Mann–Whitney test. E Simple linear regression analysis of ZOI% (invasiveness) with collagen expression showing positive correlation, P value = 0.031, R² = 0.45, r = 0.68

Fig. 3

Differential pulmosphere invasiveness in patients with ILD following exposure to nintedanib and pirfenidone. A Representative image of an invasive pulmosphere compared to pulmosphere treated with nintedanib and pirfenidone. Scale bars: 100 μm. B, C Paired scatter plots depicting change in ZOI% of untreated (black) versus nintedanib (red) ILD pulmospheres, and untreated (white) versus pirfenidone (grey) treated ILD pulmospheres. Untreated (95% CI 465.1, 516.9 ± 119.9) vs nintedanib (95% CI 165.4,218.6, 192 ± 61.5, ****P value < 0.0001 by Wilcoxon test). Untreated (95% CI 471.4, 581.2 ± 126.9) vs pirfenidone (95% CI 444.5, 580.1, 512.3 ± 156.7, P value ns by Wilcoxon test). D Measurement of fold change of zone of invasion (ZOI) was calculated as the ratio of ZOI% with treatment to ZOI% without treatment in vitro with antifibrotic drug. E Median values of ZOI > 1 represent pulmospheres nonresponsive to the treatment, and median values of ZOI < 1 represent pulmospheres responsive to drug treatment. For each patient tested, 5 pulmospheres were seeded without either test drug; 5 pulmospheres were seeded with nintedanib (1 μM); and 5 pulmospheres were seeded with pirfenidone (100 μM). ZOI fold change values were obtained from the ratio of ZOI% with treatment to ZOI% without treatment of pulmospheres in vitro with antifibrotic drug for each patient. F Dot and whisker plot for ILD patient pulmospheres treated with nintedanib (red circles) and pirfenidone (black squares). Whiskers show maximum to minimum values. G Dot and whisker plot for CTD-ILD patient pulmospheres treated with pirfenidone (black squares). Dot and whisker plot depicting response to nintedanib (H) and pirfenidone (I) among ILD pulmospheres grouped into progressors (red circle), non-progressors (blue inverted triangle), and undetermined (no follow up data, black squares)

Fig. 3

Differential pulmosphere invasiveness in patients with ILD following exposure to nintedanib and pirfenidone. A Representative image of an invasive pulmosphere compared to pulmosphere treated with nintedanib and pirfenidone. Scale bars: 100 μm. B, C Paired scatter plots depicting change in ZOI% of untreated (black) versus nintedanib (red) ILD pulmospheres, and untreated (white) versus pirfenidone (grey) treated ILD pulmospheres. Untreated (95% CI 465.1, 516.9 ± 119.9) vs nintedanib (95% CI 165.4,218.6, 192 ± 61.5, ****P value < 0.0001 by Wilcoxon test). Untreated (95% CI 471.4, 581.2 ± 126.9) vs pirfenidone (95% CI 444.5, 580.1, 512.3 ± 156.7, P value ns by Wilcoxon test). D Measurement of fold change of zone of invasion (ZOI) was calculated as the ratio of ZOI% with treatment to ZOI% without treatment in vitro with antifibrotic drug. E Median values of ZOI > 1 represent pulmospheres nonresponsive to the treatment, and median values of ZOI < 1 represent pulmospheres responsive to drug treatment. For each patient tested, 5 pulmospheres were seeded without either test drug; 5 pulmospheres were seeded with nintedanib (1 μM); and 5 pulmospheres were seeded with pirfenidone (100 μM). ZOI fold change values were obtained from the ratio of ZOI% with treatment to ZOI% without treatment of pulmospheres in vitro with antifibrotic drug for each patient. F Dot and whisker plot for ILD patient pulmospheres treated with nintedanib (red circles) and pirfenidone (black squares). Whiskers show maximum to minimum values. G Dot and whisker plot for CTD-ILD patient pulmospheres treated with pirfenidone (black squares). Dot and whisker plot depicting response to nintedanib (H) and pirfenidone (I) among ILD pulmospheres grouped into progressors (red circle), non-progressors (blue inverted triangle), and undetermined (no follow up data, black squares)

Fig. 4

A Correlation analysis of ZOI% (invasiveness) with ZOI ratio(efficacy) with nintedanib treatment does not show correlation, P value = 0.49, R² = 0.0224, r = 0.1496. B Correlation analysis of ZOI% (invasiveness) with ZOI ratio(efficacy) with pirfenidone treatment does not show correlation, P value = 0.204, R² = 0.0755, r = 0.2747. C Correlation analysis of Δ %FVC (change in %FVC) with ZOI ratio(efficacy) with nintedanib treatment does not show correlation, P value = 0.444, R² = 0.0329, r = 0.1813. D Correlation analysis of Δ %FVC (change in %FVC) with ZOI ratio (efficacy) with pirfenidone treatment does not show correlation, P value = 0.0850, R² = 0.1558, r = 0.3947