(118e) Lentiviral-Based Fluorescent Reporters for Assessing Human Lung Fibroblast Activation in Response to Microenvironmental Stimuli in Multidimensional Culture Systems

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with no cure, characterized by the uncontrolled accumulation of scar tissue in the lower regions of the lung, leading to irreversible stiffening of tissue, decreased organ function and eventual respiratory failure. One significant driver of this progression is the persistent activation of fibroblasts. Fibroblast activation is a critical process for healthy healing after tissue injury, facilitating extracellular matrix (ECM) regeneration and wound contraction. However, when this phenotype becomes persistent, it leads to excess secretion of ECM proteins and crosslinking of collagen, as well as misregulated crosstalk with other critical cell types, further driving disease progression.¹ While current model systems for IPF have provided critical insights, there is still much unknown about the mechanisms that drive initiation and progression of human fibrotic disease. Consequently, therapeutic options for IPF are limited with only a few drugs that slow progression. Robust and relevant human culture models are needed to better understand these mechanisms and to identify potential therapeutic targets and strategies for halting disease progression.

A key challenge for human model system design is the need for tools for real-time assessment of cellular responses to microenvironmental stimuli. While the dynamics of cell-microenvironment interactions are thought to be instrumental in disease progression, cell behavior assessment tools are often limited to common endpoint, destructive methods incapable of assessing these dynamics. Increasing the number of experimental timepoints for more temporal resolution often requires prohibitively high sample counts for adequate assessment. Further, fibroblast populations tend to be quite heterogeneous, and the population averages from these techniques can mask critical differences occurring within cell sub-populations. There exists a need for innovative approaches to assess temporal changes in fibroblast activation behavior, from a range of donor types, in physiologically relevant, multidimensional culture systems.

Approach and Experimental Methods:

To address this need, we have established an approach to monitor the responses of human lung fibroblasts using a lentiviral-based dual-fluorescence reporter system that allows for quantification on a single-cell basis. In this system, cells constitutively express red fluorescence and dynamically express green fluorescence conditional on expression of alpha smooth muscle actin (αSMA), a common indicator of fibroblast activation.² We have implemented three versions of this reporter system with varying green fluorescence half-lives, allowing us to elucidate multiple response timescales.

Stable reporter cell lines were produced from human lung fibroblasts derived from healthy and IPF, male and female patients for facile investigation of intrinsic differences driven by sex and disease state. Utility of the reporter system was assessed by culturing these reporter cell lines in microenvironments with well-characterized activating biochemical and mechanical stimuli. Reporter fibroblasts were cultured on top of rat tail collagen I (ColI) coated polyacrylamide hydrogels (Matrigen, E~2kPa) or ColI-coated glass-bottomed well plates (Sigma-Aldrich, CellVis) to track changes in activation over time. During culture on both substrates, fibroblasts were treated with TGFβ1, a well-characterized activating cytokine, and fluorescence expression over time was assessed on a single-cell and population basis using live-cell imaging confocal microscopy, as well as endpoint flow cytometry and immunostaining.

Results and Conclusions:

In initial assessments, reporter cell lines demonstrated marked differences in the baseline expression of αSMA between healthy and IPF tissue-derived cells, demonstrating the reporter’s ability to assess variation due to intrinsic factors. Further, when reporter cells were treated with activating stimuli (hyperphysiological stiffness, TGFβ1), they expressed increased green fluorescence intensity that correlated with αSMA expression and an activated phenotype. With this work, we have demonstrated that we can assess dynamic responses of lung fibroblast cell lines to biochemical and mechanical changes within the cell microenvironment. Future studies will allow for cell behavior investigations of both cell lines and primary patient-derived cell populations in more complex synthetic microenvironments that capture additional structural elements of diseased tissue, towards establishing a robust, representative human fibrosis culture model for understanding disease mechanisms and identifying new potential therapeutic targets.

References:

1. C. Hewlett, J.A. Kropski, and T.S. Blackwell. Matrix Biology. 71, 112-127 (2018)

2. Alimperti, P. Lei, J. Tian, and S.T. Andreadis. Gene Therapy. 19, 1123-1132 (2012)