Quantitative Immunocytochemistry (QICC)-Based Approach for Antifibrotic Drug Testing in vitro

Abstract

Aim: Chemicals that can reduce extracellular collagen accumulation and inhibit the transdifferentation of fibroblast into myofibroblasts marked by expression of α-SMA (α-smooth muscle actin) will be promising antifibrotic drugs. We developed a QICC-based approach using microplatereader to quantify the deposited collagen and α-SMA for antifibrotic drug testing. Methodology: Fibroblasts IMR-90 were cultured under standard and EVE (excluded-volume effect) conditions. "Footprints" were revealed by removing cells with detergent. TSA (trichostatin A) with or w/o TGF-β1 or CPX (ciclopirox olamine) was added under EVE conditions. Collagen I and α-SMA were labeled by immunocytochemistry. The fluorescence signal was quantified by BMG PHERAstar with a focusing lens system. Biochemical evaluation of collagen production served as a benchmark. Results: The QICC-based assay detected 7-fold enhanced collagen deposition under EVE condition and "footprints" was 40% of total collagen matrix, well comparable with that of biochemical assay. Dosedependent inhibition of TGF-β1-induced collagen production and α-SMA expression by TSA and 90% decrease of collagen production by CPX were successfully detected by QICC-based approach. Conclusion: We have shown, for the first time, the feasibility of QICC-based approach using microplate-reader to quantify collagen deposition and α-SMA in vitro, thus enabling screening for candidate drugs that are likely to prevent scarring.