Classification of differentially activated groups of fibroblasts using morphodynamic and motile features.

Fibroblasts play essential roles in cancer progression, exhibiting activation states that can either promote or inhibit tumor growth. Understanding these differential activation states is critical for targeting the tumor microenvironment (TME) in cancer therapy. However, traditional molecular markers used to identify cancer-associated fibroblasts are limited by their co-expression across multiple fibroblast subtypes, making it difficult to distinguish specific activation states. Morphological and motility characteristics of fibroblasts reflect their underlying gene expression patterns and activation states, making these features valuable descriptors of fibroblast behavior. This study proposes an artificial intelligence-based classification framework to identify and characterize differentially activated fibroblasts by analyzing their morphodynamic and motile features. We extract these features from label-free live-cell imaging data of fibroblasts co-cultured with breast cancer cell lines using deep learning and machine learning algorithms. Our findings show that morphodynamic and motile features offer robust insights into fibroblast activation states, complementing molecular markers and overcoming their limitations. This biophysical state-based cellular classification framework provides a novel, comprehensive approach for characterizing fibroblast activation, with significant potential for advancing our understanding of the TME and informing targeted cancer therapies.