(676) A multi-center approach to optimize and validate a robust CFTR-specific immunolabeling protocol

Background: Cystic Fibrosis (CF) is a monogenic disease caused by loss-of-function mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The CF Foundation’s “Path to a Cure” initiative has substantially increased the number of groups that seek to address CF with genetic therapies. No matter if the therapeutic modality is mRNA, gene replacement, or gene-editing, all the programs share the need to confidently and reproducibly detect localized expression of the CFTR protein in healthy and CF cells and tissue. To date, there is no CFTR immunolabeling protocol that has been widely adapted. Here, we report on our progress in the protocol development for CFTR immunolabeling, focusing on genetic controls and experimental formats including in vitro, e.g., cell lines, human bronchial epithelial (hBE), and human nasal epithelial cells (hNE) air-liquid interface (ALI) cultures, and ex vivo, e.g., formalin-fixed paraffin-embedded experiments.

Methods: Working collaboratively across 3 sites (CFF/UNC/Toronto) we independently performed a range of immunostaining techniques across multiple CFTR antibodies reported in the literature. Initially, we used the CFTR (wt/wt) cell line 16HBE14o- and the isogenic derivative CFF-16HBEge CFTR G542X, which is homozygous for CFTR G542X, a CFTR variant harboring a Premature Termination Codon (PTC), and has no detectable CFTR expression. Variables that were tested included fixation method, detergent, primary antibody, and visualization by direct label versus enzymatic amplification. Next, we validated the optimized protocol for detection of CFTR in control and CF well-differentiated hBE and hNE cells. CFTR expression was then confirmed by functional measurements in Ussing chambers and by CFTR Western blotting.

Results: We generated a highly specific and reproducible CFTR immunofluorescence staining protocol that can be adapted to many experimental formats. We confirmed CFTR specificity with robust CFTR signal with mAbs 596 (UNC) and 13-1 (R&D Systems) in non-CF cultures while observing only a faint if any background signal detected in the PTC variant cell lines. Similar results, although not in genetically matched backgrounds, were obtained with non-CF vs. CFTR-variant primary human airway epithelial cells differentiated at ALI. We further demonstrated CFTR expression within distinct cell lineages, e.g., ionocytes by co-staining CFTR with FOXI1 and ATP6V1B1 in non-CF hBE ALI cultures.

Conclusions: The CFTR immunostaining protocol described is CFTR-specific and can be used across experimental formats. From our tests, we believe several protocol components to be critical for success including good quality well-differentiated primary cultures, choice of antibody, fixation, and permeabilization. To date, we are confident in our staining pattern in cells that express high levels of CFTR, yet further investigation is needed to understand the lower limits of detection. A robust and standardized CFTR immunostaining protocol will provide a valuable tool for CF research and therapeutics development.

Acknowledgements:

References: