CU Cilia – An Application for Image Analysis by Machine Learning – Reveals Significance of Cysteine Cathepsin K Activity for Primary Cilia of Human Thyroid Epithelial Cells

Maren Rehders¹Kseniia Alekseitseva²Joao Gissoni¹Alara G Dogru¹Petr Popov^1,2Andrei Boiarov^2*Klaudia Brix^1*

• ¹Constructor University, Bremen, Bremen, Germany
• ²Constructor Tech, Sofia, Bulgaria

Cysteine cathepsins K and L are crucial for proper thyroid function in mice. Inhibition of cysteine peptidases in rodent thyroid epithelial cells in vitro results in loss of primary cilia, highlighting the significance of proteolysis for cilia maintenance. In this study, we used an in vitro-approach to ask whether primary cilia of the normal human thyroid epithelial cell line Nthy-ori 3-1 are affected by cysteine cathepsin inhibition in numbers and/or structure. To interpret hundreds of primary cilia in dozens of high-resolution laser-scanning microscopic images, an objective image analysis approach is essential. Therefore, we developed CU Cilia, i.e., a method for the detection of primary cilia and for segmentation of nuclei by machine learning-based image analysis algorithms. For validation of CU Cilia, the results of nuclei segmentation and primary cilia detection are compared with the image analysis software Cell Profiler™. While manual editing by an image analysis expert enhances the already very good image segmentation results when using Cell Profiler™, the herein described CU Cilia application achieved well-comparable results. CU Cilia even allows non-expert users to perform image analysis with little to no training. CU Cilia uses machine learning by a U-Net neural network simplified architecture and requires only a few images as training datasets. Both approaches, Cell Profiler™ and the CU Cilia application, revealed that the broad-spectrum cysteine peptidase inhibitor E64d and the specific cysteine cathepsin K inhibitor Odanacatib affect primary cilia structure significantly, while their numbers remained unaffected. The results point to an elongation of primary cilia in the Odanacatib-treated Nthy-ori 3-1 thyroid epithelial cells, supporting the notion that cysteine cathepsin K activity is physiologically important not only in murine but also in human thyroid cells.